Modified adenoviruses

ABSTRACT

Compositions include modified adenoviruses. Nucleotides, cells, and methods associated with the compositions, including their use as vaccines. Viral vectors using a TET promoter system and methods of producing viruses having the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation U.S. application Ser. No. 17/538,716filed Nov. 30, 2021, which is continuation of International ApplicationNo. PCT/US2020/035591 filed Jun. 1, 2020, which application claims thebenefit of U.S. Provisional Application No. 62/854,865 filed May 30,2019, each of which is hereby incorporated by reference in its entiretyfor all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jan. 26, 2023, isnamed GSO-033C2.xml and is 800,821 bytes in size.

BACKGROUND

Therapeutic vaccines based on tumor-specific antigens hold great promiseas a next-generation of personalized cancer immunotherapy.¹⁻³ Forexample, cancers with a high mutational burden, such as non-small celllung cancer (NSCLC) and melanoma, are particularly attractive targets ofsuch therapy given the relatively greater likelihood of neoantigengeneration.^(4,5) Early evidence shows that neoantigen-based vaccinationcan elicit T-cell responses⁶ and that neoantigen targeted cell-therapycan cause tumor regression under certain circumstances in selectedpatients.⁷

One question for antigen vaccine design in both cancer and infectiousdisease settings is which of the many coding mutations present generatethe “best” therapeutic antigens, e.g., antigens that can elicitimmunity.

In addition to the challenges of current antigen prediction methodscertain challenges also exist with the available vector systems that canbe used for antigen delivery in humans, many of which are derived fromhumans. For example, many humans have pre-existing immunity to humanviruses as a result of previous natural exposure, and this immunity canbe a major obstacle to the use of recombinant human viruses for antigendelivery in vaccination strategies, such as in cancer treatment orvaccinations against infectious diseases. While some progress has beenmade in vaccinations strategies addressing the above problems,improvements are still needed, particularly for clinical applications,such as improved vaccine potency and efficacy.

SUMMARY

An adenovirus vector comprising: an adenoviral backbone comprising oneor more genes or regulatory sequences of an adenovirus genome, andwherein the adenoviral backbone comprises a partially deleted E4 genewith reference to the adenovirus genome, wherein the partially deletedE4 gene comprises a deleted or partially-deleted E4orf2 region and adeleted or partially-deleted E4orf3 region, and optionally a deleted orpartially-deleted E4orf4 region, and optionally, wherein the adenovirusvector further comprises a cassette, the cassette comprising: (1) atleast one payload nucleic acid sequence, optionally wherein the at leastone payload nucleic acid sequence encodes a polypeptide, optionallywherein the polypeptide comprises an antigen, optionally wherein theantigen comprises: a MHC class I epitope, a MHC class II epitope, anepitope capable of stimulating a B cell response, or a combinationthereof, and optionally wherein the at least one payload nucleic acidsequence further comprises a 5′ linker sequence and/or a 3′ linkersequence, and optionally wherein; (2) at least one promoter sequenceoperably linked to the at least one payload nucleic acid sequence, (3)optionally, at least one universal MHC class II antigen-encoding nucleicacid sequence; (4) optionally, at least one GPGPG-encoding linkersequence (SEQ ID NO:56); and (5) optionally, at least onepolyadenylation sequence.

Also disclosed herein is a chimpanzee adenovirus vector comprising amodified ChAdV68 sequence, wherein the modified ChAdV68 sequencecomprises: (a) a partially deleted E4 gene of the E4 gene sequence shownin SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 ofthe sequence shown in SEQ ID NO:1; and (b) one or more genes orregulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1,optionally wherein the one or more genes or regulatory sequencescomprise at least one of the chimpanzee adenovirus inverted terminalrepeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genesof the sequence shown in SEQ ID NO: 1; and optionally, wherein thechimpanzee adenovirus vector further comprises a cassette, wherein thecassette comprises at least one payload nucleic acid sequence, andwherein the cassette comprises at least one promoter sequence operablylinked to the at least one payload nucleic acid sequence.

Also disclosed herein is a chimpanzee adenovirus vector comprising amodified ChAdV68 sequence, wherein the modified ChAdV68 sequencecomprises: (a) a partially deleted E4 gene of the E4 gene sequence shownin SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 ofthe sequence shown in SEQ ID NO:1; (b) nucleotides 2 to 34,916 of thesequence shown in SEQ ID NO:1, wherein the partially deleted E4 gene is3′ of the nucleotides 2 to 34,916, and optionally the nucleotides 2 to34,916 additionally lack nucleotides 577 to 3403 of the sequence shownin SEQ ID NO:1 corresponding to an E1 deletion and/or lack nucleotides27,125 to 31,825 of the sequence shown in SEQ ID NO:1 corresponding toan E3 deletion; and (c) nucleotides 35,643 to 36,518 of the sequenceshown in SEQ ID NO:1, and wherein the partially deleted E4 gene is 5′ ofthe nucleotides 35,643 to 36,518, and optionally, wherein the chimpanzeeadenovirus vector further comprises a cassette, wherein the cassettecomprises at least one payload nucleic acid sequence, and wherein thecassette comprises at least one promoter sequence operably linked to theat least one payload nucleic acid sequence.

Also disclosed herein is a chimpanzee adenovirus vector comprising: a. amodified ChAdV68 sequence, wherein the modified ChAdV68 sequencecomprises: (i) a partially deleted E4 gene of the E4 gene sequence shownin SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 ofthe sequence shown in SEQ ID NO:1; (ii) nucleotides 2 to 34,916 of thesequence shown in SEQ ID NO:1, wherein the partially deleted E4 gene is3′ of the nucleotides 2 to 34,916, and optionally the nucleotides 2 to34,916 additionally lack nucleotides 577 to 3403 of the sequence shownin SEQ ID NO:1 corresponding to an E1 deletion and/or lack nucleotides27,125 to 31,825 of the sequence shown in SEQ ID NO:1 corresponding toan E3 deletion; and (iii) and nucleotides 35,643 to 36,518 of thesequence shown in SEQ ID NO:1, and wherein the partially deleted E4 geneis 5′ of the nucleotides 35,643 to 36,518, and; b. a CMV-derivedpromoter sequence; c. an SV40 polyadenylation signal nucleotidesequence; and d. a cassette, the cassette comprising at least one atleast one payload nucleic acid sequence encoding: at least one MHC classI epitope, optionally wherein the at least one MHC class I epitopecomprises at least 2 distinct MHC class I epitopes linearly linked toeach other and each optionally comprising: (A) at least one alterationthat makes the encoded peptide sequence distinct from the correspondingpeptide sequence encoded by a wild-type nucleic acid sequence, whereinthe distinct MHC I epitope is 7-15 amino acids in length, (B) anN-terminal linker comprising a native N-terminal amino acid sequence ofthe distinct MHC I epitope that is at least 3 amino acids in length, (C)an C-terminal linker comprising a native C-terminal acid sequence of thedistinct MHC I epitope that is at least 3 amino acids in length, or (D)combinations thereof, at least one MHC class II epitope, optionallywherein the at least one MHC class II epitope comprises at least 2distinct MHC class II epitopes, at least one an epitope capable ofstimulating a B cell response, or combinations thereof, and wherein thecassette is inserted within a deleted region of ChAdV68 and theCMV-derived promoter sequence is operably linked to the cassette.

Also disclosed herein is a method for stimulating an immune response ina subject, the method comprising administering to the subject anadenovirus vector comprising: an adenoviral backbone comprising one ormore genes or regulatory sequences of an adenovirus genome, and whereinthe adenoviral backbone comprises a partially deleted E4 gene withreference to the adenovirus genome, wherein the partially deleted E4gene comprises a deleted or partially-deleted E4orf2 region and adeleted or partially-deleted E4orf3 region, and optionally a deleted orpartially-deleted E4orf4 region, and wherein the adenovirus vectorfurther comprises a cassette, the cassette comprising: (1) at least onepayload nucleic acid sequence, optionally wherein the at least onepayload nucleic acid sequence encodes a polypeptide, optionally whereinthe polypeptide comprises an antigen, optionally wherein the antigencomprises: a MHC class I epitope, a MHC class II epitope, an epitopecapable of stimulating a B cell response, or a combination thereof, andoptionally wherein the at least one payload nucleic acid sequencefurther comprises a 5′ linker sequence and/or a 3′ linker sequence, andoptionally wherein; (2) at least one promoter sequence operably linkedto the at least one payload nucleic acid sequence, (3) optionally, atleast one universal MHC class II antigen-encoding nucleic acid sequence;(4) optionally, at least one GPGPG-encoding linker sequence (SEQ IDNO:56); and (5) optionally, at least one polyadenylation sequence.

Also disclosed herein is a method for treating a subject with a disease,optionally wherein the disease is cancer or an infection, the methodcomprising administering to the subject an adenovirus vector comprising:an adenoviral backbone comprising one or more genes or regulatorysequences of an adenovirus genome, and wherein the adenoviral backbonecomprises a partially deleted E4 gene with reference to the adenovirusgenome, wherein the partially deleted E4 gene comprises a deleted orpartially-deleted E4orf2 region and a deleted or partially-deletedE4orf3 region, and optionally a deleted or partially-deleted E4orf4region, and wherein the adenovirus vector further comprises a cassette,the cassette comprising: (1) at least one payload nucleic acid sequence,optionally wherein the at least one payload nucleic acid sequenceencodes a polypeptide, optionally wherein the polypeptide comprises anantigen, optionally wherein the antigen comprises: a MHC class Iepitope, a MHC class II epitope, an epitope capable of stimulating a Bcell response, or a combination thereof, and optionally wherein the atleast one payload nucleic acid sequence further comprises a 5′ linkersequence and/or a 3′ linker sequence, and optionally wherein; (2) atleast one promoter sequence operably linked to the at least one payloadnucleic acid sequence, (3) optionally, at least one universal MHC classII antigen-encoding nucleic acid sequence; (4) optionally, at least oneGPGPG-encoding linker sequence (SEQ ID NO:56); and (5) optionally, atleast one polyadenylation sequence.

Also disclosed herein is a method for stimulating an immune response ina subject, the method comprising administering to the subject anadenovirus vector comprising a modified ChAdV68 sequence, wherein themodified ChAdV68 sequence comprises: (a) a partially deleted E4 gene ofthe E4 gene sequence shown in SEQ ID NO:1 and that lacks at leastnucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1; and(b) one or more genes or regulatory sequences of the ChAdV68 sequenceshown in SEQ ID NO: 1, optionally wherein the one or more genes orregulatory sequences comprise at least one of the chimpanzee adenovirusinverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3,L4, and L5 genes of the sequence shown in SEQ ID NO: 1; and wherein thechimpanzee adenovirus vector further comprises a cassette, wherein thecassette comprises at least one payload nucleic acid sequence, andwherein the cassette comprises at least one promoter sequence operablylinked to the at least one payload nucleic acid sequence.

Also disclosed herein is a method for treating a subject with a disease,optionally wherein the disease is cancer or an infection, the methodcomprising administering to the subject an adenovirus vector comprisinga modified ChAdV68 sequence, wherein the modified ChAdV68 sequencecomprises: (a) a partially deleted E4 gene of the E4 gene sequence shownin SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 ofthe sequence shown in SEQ ID NO:1; and (b) one or more genes orregulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1,optionally wherein the one or more genes or regulatory sequencescomprise at least one of the chimpanzee adenovirus inverted terminalrepeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genesof the sequence shown in SEQ ID NO: 1; and wherein the chimpanzeeadenovirus vector further comprises a cassette, wherein the cassettecomprises at least one payload nucleic acid sequence, and wherein thecassette comprises at least one promoter sequence operably linked to theat least one payload nucleic acid sequence.

Also disclosed herein is a method of producing a virus, wherein themethod comprises the steps of: a. providing a viral vector comprising acassette, the cassette comprising: (i) at least one payload nucleic acidsequence, optionally wherein the at least one payload nucleic acidsequence encodes a polypeptide, optionally wherein the polypeptidecomprises an antigen, optionally wherein the antigen comprises: a MHCclass I epitope, a MHC class II epitope, an epitope capable ofstimulating a B cell response, or a combination thereof, and optionallywherein the at least one payload nucleic acid sequence further comprisesa 5′ linker sequence and/or a 3′ linker sequence, and optionallywherein; (ii) at least one promoter sequence operably linked to the atleast one payload nucleic acid sequence, wherein the at least onepromoter is a tetracycline (TET) repressor protein (TETr) controlledpromoter, (iii) optionally, at least one MHC class II antigen-encodingnucleic acid sequence; (iv) optionally, at least one GPGPG-encodinglinker sequence (SEQ ID NO:56); and (v) optionally, at least onepolyadenylation sequence; b. providing a cell engineered to express theTETr protein; and c. contacting the viral vector with the cell underconditions sufficient for production of the virus.

In some aspects, the viral vector comprises a chimpanzee adenovirusvector, optionally wherein the chimpanzee adenovirus vector is a ChAdV68vector. In some aspects, the production of the virus is increased usingthe vector comprising the TETr controlled promoter relative toproduction of a virus produced using a vector without the TETrcontrolled promoter. In some aspects, the increased production isincreased at least 1.5-fold, at least 2-fold, at least 2.5-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, or at least 10-fold relative toproduction using a vector without the TETr controlled promoter. In someaspects, the increased production is increased at least 15-fold, atleast 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, atleast 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, atleast 80-fold, at least 90-fold, or at least 100-fold relative toproduction using a vector without the TETr controlled promoter. In someaspects, the production of the virus is increased using the vectorcomprising the TETr controlled promoter relative to production of avirus produced using a cell that is not engineered to express the TETrprotein. In some aspects, the increased production is increased at least1.5-fold, at least 2-fold, at least 2.5-fold, at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least9-fold, or at least 10-fold relative to production using a cell that isnot engineered to express the TETr protein.

Also provided herein is a viral vector comprising a cassette, thecassette comprising: (i) at least one payload nucleic acid sequence,optionally wherein the at least one payload nucleic acid sequenceencodes a polypeptide, optionally wherein the polypeptide comprises anantigen, optionally wherein the antigen comprises: a MHC class Iepitope, a MHC class II epitope, an epitope capable of stimulating a Bcell response, or a combination thereof, and optionally wherein the atleast one payload nucleic acid sequence further comprises a 5′ linkersequence and/or a 3′ linker sequence, and optionally wherein; (ii) atleast one promoter sequence operably linked to the at least one payloadnucleic acid sequence, wherein the at least one promoter is atetracycline (TET) repressor protein (TETr) controlled promoter, (iii)optionally, at least one MHC class II antigen-encoding nucleic acidsequence; (iv) optionally, at least one GPGPG-encoding linker sequence(SEQ ID NO:56); and (v) optionally, at least one polyadenylationsequence.

In some aspects, the TETr controlled promoter comprises one or more TEToperator (TETo) nucleic acid sequences, optionally wherein the one ormore TETo nucleic acid sequences comprises the nucleotide sequence shownin SEQ ID NO:60. In some aspects, the one or more TETo nucleic acidsequences comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more TETo nucleicacid sequences, optionally wherein each of TETo nucleic acid sequencescomprises the nucleotide sequence shown in SEQ ID NO:60. In someaspects, the 2 or more TETo nucleic acid sequences are linked together.In some aspects, the 2 or more TETo nucleic acid sequences are directlylinked together. In some aspects, the 2 or more TETo nucleic acidsequences are linked together with a linker sequence, wherein the linkercomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 or more nucleotides, and optionally wherein the linkersequence comprises the linker nucleotide sequence shown in SEQ ID NO:61.In some aspects, the one or more TETo nucleic acid sequences are 5′ of aRNA polymerase binding sequence of the promoter sequence. In someaspects, the one or more TETo nucleic acid sequences are 3′ of a RNApolymerase binding sequence of the promoter sequence. In some aspects,the at least one promoter sequence comprises a CMV, SV40, EF-1, RSV,PGK, HSA, MCK or EBV promoter sequence. In some aspects, the at leastone promoter sequence is a CMV-derived promoter sequence, optionallywherein the CMV promoter sequence comprises the CMV promoter nucleotidesequence shown in SEQ ID NO:64. In some aspects, the CMV-derivedpromoter sequence is a minimal CMV promoter sequence, optionally whereinthe minimal CMV promoter sequence comprises the minimal CMV promoternucleotide sequence as shown in SEQ ID NO:61.

In some aspects, the TETr controlled promoter operably linked to the atleast one payload nucleic acid sequence comprises an ordered sequencedescribed in the formula, from 5′ to 3′, comprising: (T-L_(Y))_(X)-P—Nwherein, N comprises one of the at least one payload nucleic acidsequences, optionally wherein each N encodes a MHC class I epitope, aMHC class II epitope, an epitope capable of stimulating a B cellresponse, or a combination thereof, optionally with at least onealteration that makes the encoded epitope sequence distinct from thecorresponding peptide sequence encoded by a wild-type nucleic acidsequence P a RNA polymerase binding sequence of the promoter sequenceoperably linked to at least one of the at least one payload nucleic acidsequences, T comprises a TETo nucleic acid sequences comprising thenucleotide sequence shown in SEQ ID NO:60, L comprises a linkersequence, where Y=0 or 1 for each X, and wherein X=1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some aspects,the TETr controlled promoter operably linked to the at least one payloadnucleic acid sequence comprises an ordered sequence described in theformula, from 5′ to 3′, comprising: P-(T-L_(Y))_(X)-N wherein, Ncomprises one of the at least one payload nucleic acid sequences,optionally wherein each N encodes a MHC class I epitope, a MHC class IIepitope, an epitope capable of stimulating a B cell response, or acombination thereof, optionally with at least one alteration that makesthe encoded epitope sequence distinct from the corresponding peptidesequence encoded by a wild-type nucleic acid sequence P a RNA polymerasebinding sequence of the promoter sequence operably linked to at leastone of the at least one payload nucleic acid sequences, T comprises aTETo nucleic acid sequences comprising the nucleotide sequence shown inSEQ ID NO:60, L comprises a linker sequence, where Y=0 or 1 for each X,and wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20.

In some aspects, the TETr controlled promoter comprises: (1) a minimalCMV promoter sequence; (2) 7 TETo nucleic acid sequences, wherein eachof TETo nucleic acid sequences comprises the nucleotide sequence shownin SEQ ID NO:60, and wherein each of the TETo nucleic acid sequences arelinked together with a linker sequence, the 7 TETo nucleic acidsequences are 5′ of the minimal CMV promoter sequence, and optionallywherein the TETr controlled promoter comprises the nucleotide sequenceas shown in SEQ ID NO:61. In some aspects, the TETr controlled promotercomprises: (1) a CMV promoter sequence; (2) 2 TETo nucleic acidsequences, wherein each of the TETo nucleic acid sequences comprises thenucleotide sequence shown in SEQ ID NO:60, and wherein each of the TETonucleic acid sequences are directly linked together, the 2 TETo nucleicacid sequences are 3′ of the CMV promoter sequence, and optionallywherein the TETr controlled promoter comprises the nucleotide sequenceas shown in SEQ ID NO:64.

In some aspects, the viral vector comprises a vector backbone, whereinthe vector backbone comprises a chimpanzee adenovirus vector, optionallywherein the chimpanzee adenovirus vector is a ChAdV68 vector.

In some aspects, the cassette comprises an ordered sequence described inthe formula, from 5′ to 3′, comprising:P_(a)-(L5_(b)-N_(c)-L3_(d))_(X)-(G5_(e)-U_(f))_(Y)-G3_(g)-A_(h) wherein,N comprises one of the at least one payload nucleic acid sequences,optionally wherein each N encodes a MHC class I epitope, a MHC class IIepitope, an epitope capable of stimulating a B cell response, or acombination thereof, optionally with at least one alteration that makesthe encoded epitope sequence distinct from the corresponding peptidesequence encoded by a wild-type nucleic acid sequence, where c=1, Pcomprises the at least one promoter sequence operably linked to at leastone of the at least one payload nucleic acid sequences, where a=1, L5comprises the 5′ linker sequence, where b=0 or 1, L3 comprises the 3′linker sequence, where d=0 or 1, G5 comprises one of the at least onenucleic acid sequences encoding a GPGPG amino acid linker (SEQ ID NO:56), where e=0 or 1, G3 comprises one of the at least one nucleic acidsequences encoding a GPGPG amino acid linker (SEQ ID NO: 56), where g=0or 1, U comprises one of the at least one universal MHC class IIantigen-encoding nucleic acid sequence, where f=1, A comprises the atleast one polyadenylation sequence, where h=0 or 1, X=2 to 400, wherefor each X the corresponding N_(c) is a payload nucleic acid sequence,optionally wherein for each X the corresponding N_(c) is a distinctpayload nucleic acid sequence, and Y=0-2, where for each Y thecorresponding U_(f) is a universal MHC class II antigen-encoding nucleicacid sequence, optionally wherein for each Y the corresponding U_(f) isa distinct universal MHC class II antigen-encoding nucleic acidsequence.

In some aspects, the cassette further comprises at least one additionalpayload nucleic acid sequence not encoded in the ordered sequence. Insome aspects, b=1, d=1, e=1, g=1, h=1, X=10, Y=2, P is a CMV-derivedpromoter sequence, each N encodes a MHC class I epitope, a MHC class IIepitope, an epitope capable of stimulating a B cell response, or acombination thereof, L5 encodes a native N-terminal amino acid sequenceof the epitope, and wherein the 5′ linker sequence encodes a peptidethat is at least 3 amino acids in length, L3 encodes a native C-terminalamino acid sequence of the epitope, and wherein the 3′ linker sequenceencodes a peptide that is at least 3 amino acids in length, U is each ofa PADRE class II sequence and a Tetanus toxoid MHC class II sequence,and the vector comprises a modified ChAdV68 sequence, wherein themodified ChAdV68 sequence comprises: (a) a partially deleted E4 gene ofthe E4 gene sequence shown in SEQ ID NO:1 and that lacks at leastnucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1; (b)nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO:1, whereinthe partially deleted E4 gene is 3′ of the nucleotides 2 to 34,916, andoptionally the nucleotides 2 to 34,916 additionally lack nucleotides 577to 3403 of the sequence shown in SEQ ID NO:1 corresponding to an E1deletion and/or lack nucleotides 27,125 to 31,825 of the sequence shownin SEQ ID NO:1 corresponding to an E3 deletion; and (c) nucleotides35,643 to 36,518 of the sequence shown in SEQ ID NO:1, and wherein thepartially deleted E4 gene is 5′ of the nucleotides 35,643 to 36,518.

In some aspects, the vector is a chimpanzee adenovirus vector,optionally wherein the chimpanzee adenovirus vector is a ChAdV68 vector.

In some aspects, the partially deleted E4 gene comprises: A. the E4 genesequence shown in SEQ ID NO:1 and that lacks at least nucleotides 34,916to 35,642 of the sequence shown in SEQ ID NO:1, B. the E4 gene sequenceshown in SEQ ID NO:1 and that lacks at least nucleotides 34,916 to34,942, nucleotides 34,952 to 35,305 of the sequence shown in SEQ IDNO:1, nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO:1,and wherein the vector comprises at least nucleotides 2 to 36,518 of thesequence shown in SEQ ID NO:1, C. the E4 gene sequence shown in SEQ IDNO:1 and that lacks at least nucleotides 34,980 to 36,516 of thesequence shown in SEQ ID NO:1, and wherein the vector comprises at leastnucleotides 2 to 36,518 of the sequence shown in SEQ ID NO:1, D. the E4gene sequence shown in SEQ ID NO:1 and that lacks at least nucleotides34,979 to 35,642 of the sequence shown in SEQ ID NO:1, and wherein thevector comprises at least nucleotides 2 to 36,518 of the sequence shownin SEQ ID NO:1, E. an E4 deletion of at least a partial deletion ofE4Orf2, a fully deleted E4Orf3, and at least a partial deletion ofE4Orf4, F. an E4 deletion of at least a partial deletion of E4Orf2, atleast a partial deletion of E4Orf3, and at least a partial deletion ofE4Orf4, G. an E4 deletion of at least a partial deletion of E4Orf1, afully deleted E4Orf2, and at least a partial deletion of E4Orf3, or H.an E4 deletion of at least a partial deletion of E4Orf2 and at least apartial deletion of E4Orf3.

In some aspects, the vector comprises one or more genes or regulatorysequences of the ChAdV68 sequence shown in SEQ ID NO: 1, optionallywherein the one or more genes or regulatory sequences are selected fromthe group consisting of the chimpanzee adenovirus inverted terminalrepeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genesof the sequence shown in SEQ ID NO: 1. In some aspects, the adenoviralbackbone or modified ChAdV68 sequence further comprises a functionaldeletion in at least one gene selected from the group consisting of anadenovirus E1A, E1B, E2A, E2B, E3, L1, L2, L3, L4, and L5 gene withreference to the adenovirus genome or with reference to the sequenceshown in SEQ ID NO: 1, optionally wherein the adenoviral backbone ormodified ChAdV68 sequence is fully deleted or functionally deleted in:(1) E1A and E1B; or (2) E1A, E1B, and E3 with reference to theadenovirus genome or with reference to the sequence shown in SEQ ID NO:1, optionally wherein the E1 gene is functionally deleted through an E1deletion of at least nucleotides 577 to 3403 with reference to thesequence shown in SEQ ID NO: 1 and optionally wherein the E3 gene isfunctionally deleted through an E3 deletion of at least nucleotides27,125 to 31,825 with reference to the sequence shown in SEQ ID NO: 1.

In some aspects, the cassette is present and is inserted in the vectorat the E1 region, E3 region, and/or any deleted AdV region that allowsincorporation of the cassette.

In some aspects, the vector is generated from one of a first generation,a second generation, or a helper-dependent adenoviral vector.

In some aspects, the modified ChAdV68 sequence comprises nucleotides 2to 34,916 of the sequence shown in SEQ ID NO:1, wherein the partiallydeleted E4 gene is 3′ of the nucleotides 2 to 34,916. In some aspects,the nucleotides 2 to 34,916 lack nucleotides 577 to 3403 of the sequenceshown in SEQ ID NO:1 corresponding to an E1 deletion. In some aspects,the nucleotides 2 to 34,916 lack nucleotides 456-3014 with reference tothe sequence shown in SEQ ID NO: 1. In some aspects, the nucleotides 2to 34,916 lack nucleotides 27,125-31,825 with reference to the sequenceshown in SEQ ID NO:1 corresponding to an E3 deletion. In some aspects,the nucleotides 2 to 34,916 lack nucleotides 27,816-31,333 withreference to the sequence shown in SEQ ID NO:1. In some aspects, thenucleotides 2 to 34,916 lack nucleotides 577 to 3403 of the sequenceshown in SEQ ID NO:1 corresponding to an E1 deletion and lacknucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1corresponding to an E3 deletion. In some aspects, the nucleotides 2 to34,916 further lack nucleotides 3957-10346, nucleotides 21787-23370,nucleotides 33486-36193, or a combination thereof with reference to thesequence shown in SEQ ID NO: 1.

In some aspects, at least one of the at least one payload nucleic acidsequences encodes an antigen, wherein the antigen comprises: a MHC classI epitope, a MHC class II epitope, an epitope capable of stimulating a Bcell response, or a combination thereof. In some aspects, at least oneof the at least one payload nucleic acid sequences encodes a polypeptidesequence capable of undergoing antigen processing into an epitope,optionally wherein the epitope is known or suspected to be presented byMHC class I on a surface of a cell, optionally wherein the surface ofthe cell is a tumor cell surface or an infected cell surface.

In some aspects, at least one of the at least one payload nucleic acidsequences encodes a polypeptide sequence or portion thereof that ispresented by MHC class I and/or MHC class II on a surface of a cell,optionally wherein the surface of the cell is a tumor cell surface or aninfected cell surface. In some aspects, the a tumor cell selected fromthe group consisting of: lung cancer, melanoma, breast cancer, ovariancancer, prostate cancer, kidney cancer, gastric cancer, colon cancer,testicular cancer, head and neck cancer, pancreatic cancer, braincancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenousleukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia,non-small cell lung cancer, and small cell lung cancer, or the infectedcell selected from the group consisting of: a pathogen infected cell, avirally infected cell, a bacterially infected cell, an fungally infectedcell, and a parasitically infected cell, optionally wherein the virallyinfected cell is selected from the group consisting of: an HIV infectedcell, a Severe acute respiratory syndrome-related coronavirus (SARS)infected cell, a severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infected cell, a Ebola infected cell, a Hepatitis B virus(HBV) infected cell, an influenza infected cell, and a Hepatitis C virus(HCV) infected cell.

In some aspects, at least one of the at least one payload nucleic acidsequences encodes a polypeptide sequence or portion thereof comprisingan epitope capable of stimulating a B cell response, optionally whereinthe polypeptide sequence or portion thereof comprises a full-lengthprotein, a protein domain, a protein subunit, or an antigenic fragmentpredicted or known to be capable of being bound by an antibody.

In some aspects, at least one of the at least one payload nucleic acidsequences encodes an infectious disease organism peptide selected fromthe group consisting of: a pathogen-derived peptide, a virus-derivedpeptide, a bacteria-derived peptide, a fungus-derived peptide, and aparasite-derived peptide. In some aspects, at least one of the at leastone payload nucleic acid sequences encodes an epitope with at least onealteration that makes the encoded epitope sequence distinct from thecorresponding peptide sequence encoded by a wild-type nucleic acidsequence. In some aspects, at least one of the at least one payloadnucleic acid sequences encodes a MHC class I epitope or MHC class IIepitope with at least one alteration that makes the encoded peptidesequence distinct from the corresponding peptide sequence encoded by awild-type nucleic acid sequence, optionally wherein the encodedpolypeptide sequence or portion thereof has increased binding affinityto, increased binding stability to, and/or an increased likelihood ofpresentation on its corresponding MHC allele relative to the translated,corresponding wild-type nucleic acid sequence. In some aspects, the atleast one alteration comprises a point mutation, a frameshift mutation,a non-frameshift mutation, a deletion mutation, an insertion mutation, asplice variant, a genomic rearrangement, or a proteasome-generatedspliced antigen.

In some aspects, at least one of the at least one payload nucleic acidsequences encodes a full-length protein, a protein domain, or a proteinsubunit. In some aspects, at least one of the at least one payloadnucleic acid sequences encodes an antibody, a cytokine, a chimericantigen receptor (CAR), a T-cell receptor, and a genome-editing systemnuclease.

In some aspects, at least one of the at least one payload nucleic acidsequences comprises a non-coding nucleic acid sequence. In some aspects,the non-coding nucleic acid sequence comprises an RNA interference(RNAi) polynucleotide or genome-editing system polynucleotide.

In some aspects, each of the at least one payload nucleic acid sequencesis linked directly to one another. In some aspects, at least one of theat least one payload nucleic acid sequences is linked to a distinctpayload nucleic acid sequence with a nucleic acid sequence encoding alinker. In some aspects, the linker links two payload nucleic acidsequences encoding MHC class I epitopes or links a first payload nucleicacid sequence encoding an MHC class I epitope to a second payloadnucleic acid sequence encoding an MHC class II epitope or encoding anepitope sequence capable of stimulating a B cell response. In someaspects, the linker is selected from the group consisting of: (1)consecutive glycine residues, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10residues in length; (2) consecutive alanine residues, at least 2, 3, 4,5, 6, 7, 8, 9, or 10 residues in length; (3) two arginine residues (RR);(4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that isprocessed efficiently by a mammalian proteasome; and (6) one or morenative sequences flanking the antigen derived from the cognate proteinof origin and that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. Insome aspects, the linker links two payload nucleic acid sequencesencoding MHC class II epitopes or links a first payload nucleic acidsequence encoding an MHC class II epitope to a second payload nucleicacid sequence encoding an MHC class I epitope or encoding an epitopesequence capable of stimulating a B cell response. In some aspects, thelinker comprises the sequence GPGPG (SEQ ID NO: 56).

In some aspects, at least one of the at least one payload nucleic acidsequences is linked, operably or directly, to a separate or contiguoussequence that enhances the expression, stability, cell trafficking,processing and presentation, and/or immunogenicity of the at least onepayload nucleic acid sequence, and optionally the expression, stability,cell trafficking, processing and presentation, and/or immunogenicity ofthe polypeptide encoded by the at least one payload nucleic acidsequence. In some aspects, the separate or contiguous sequence comprisesat least one of: a ubiquitin sequence, a ubiquitin sequence modified toincrease proteasome targeting, optionally wherein the ubiquitin sequencecontains a Gly to Ala substitution at position 76, an immunoglobulinsignal sequence, optionally wherein the immunoglobulin signal sequencecomprises IgK, a major histocompatibility class I sequence,lysosomal-associated membrane protein (LAMP)-1, human dendritic celllysosomal-associated membrane protein, and a major histocompatibilityclass II sequence; optionally wherein the ubiquitin sequence modified toincrease proteasome targeting is A76.

In some aspects, the expression of each of the at least one payloadnucleic acid sequences is driven by the at least one promoter.

In some aspects, the at least one payload nucleic acid sequencecomprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 payload nucleic acidsequences. In some aspects, the at least one payload nucleic acidsequence comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or upto 400 payload nucleic acid sequences. In some aspects, the at least onepayload nucleic acid sequence comprises at least 2-400 payload nucleicacid sequences and wherein at least one of the at least one payloadnucleic acid sequences encodes a MHC class I epitope, a MHC class IIepitope, an epitope capable of stimulating a B cell response, or acombination thereof. In some aspects, the at least one payload nucleicacid sequence comprises at least 2-400 payload nucleic acid sequencesand wherein, when administered to the subject and translated, at leastone of the at least one payload nucleic acid sequences encodes anantigen presented on antigen presenting cells resulting in an immuneresponse targeting the antigen. In some aspects, the at least onepayload nucleic acid sequence comprises at least 2-400 MHC class Iand/or class II antigen-encoding nucleic acid sequences, wherein, whenadministered to the subject and translated, at least one of the MHCclass I or class II antigens are presented on antigen presenting cellsresulting in an immune response targeting at least one of the antigenson a cell surface, and optionally wherein the expression of each of theat least 2-400 MHC class I or class II antigen-encoding nucleic acidsequences is driven by the at least one promoter.

In some aspects, each MHC class I epitope is independently between 8 and35 amino acids in length, optionally 7-15, 9-17, 9-25, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34 or 35 amino acids in length. In some aspects, the atleast one universal MHC class II antigen-encoding nucleic acid sequenceis present. In some aspects, the at least one universal MHC class IIantigen-encoding nucleic acid sequence is present and comprises at leastone universal MHC class II antigen-encoding nucleic acid sequence thatcomprises at least one alteration that makes the encoded peptidesequence distinct from the corresponding peptide sequence encoded by awild-type nucleic acid sequence. In some aspects, the at least oneuniversal MHC class II antigen-encoding nucleic acid sequence is 12-20,12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. Insome aspects, the at least one universal MHC class II antigen-encodingnucleic acid sequence is present and wherein the at least one universalsequence comprises at least one of Tetanus toxoid and PADRE.

In some aspects, the at least one promoter sequence is a regulatablepromoter, optionally wherein the regulatable promoter is a tetracycline(TET) repressor protein (TETr) controlled promoter, optionally whereinthe regulatable promoter comprises multiple TET operator (TETo)sequences 5′ or 3′ of a RNA polymerase binding sequence of the promoter.multiple TET operator (TETo) sequences are 5′ or 3′ of a RNA the atleast one promoter sequence is constitutive. multiple TET operator(TETo) sequences are 5′ or 3′ of a RNA the at least one promotersequence is a CMV, SV40, EF-1, RSV, PGK, HSA, MCK or EBV promotersequence.

In some aspects, the cassette further comprises at least onepoly-adenylation (polyA) sequence operably linked to at least one of theat least one payload nucleic acid sequences, optionally wherein thepolyA sequence is located 3′ of the at least one payload nucleic acidsequence. In some aspects, the polyA sequence comprises an SV40 orBovine Growth Hormone (BGH) polyA sequence.

In some aspects, the cassette further comprises at least one of: anintron sequence, a woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE) sequence, an internal ribosome entry sequence(IRES) sequence, a nucleotide sequence encoding a 2A self-cleavingpeptide sequence, a nucleotide sequence encoding a Furin cleavage site,a nucleotide sequence encoding a TEV cleavage site, or a sequence in the5′ or 3′ non-coding region known to enhance the nuclear export,stability, or translation efficiency of mRNA that is operably linked toat least one of the at least one payload nucleic acid sequences.

In some aspects, the cassette comprises a reporter gene, including butnot limited to, green fluorescent protein (GFP), a GFP variant, secretedalkaline phosphatase, luciferase, or a luciferase variant.

In some aspects, the vector further comprises one or more payloadnucleic acid sequences encoding at least one immune modulator,optionally wherein the at least one immune modulator inhibits an immunecheckpoint molecule. In some aspects, the immune modulator is ananti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD-1antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibodyor an antigen-binding fragment thereof, an anti-4-1BB antibody or anantigen-binding fragment thereof, or an anti-OX-40 antibody or anantigen-binding fragment thereof. In some aspects, the antibody orantigen-binding fragment thereof is a Fab fragment, a Fab′ fragment, asingle chain Fv (scFv), a single domain antibody (sdAb) either as singlespecific or multiple specificities linked together (e.g., camelidantibody domains), or full-length single-chain antibody (e.g.,full-length IgG with heavy and light chains linked by a flexiblelinker). In some aspects, the heavy and light chain sequences of theantibody are a contiguous sequence separated by either a self-cleavingsequence such as 2A, optionally wherein the self-cleaving sequence has aFurin cleavage site sequence 5′ of the self-cleaving sequence, or anIRES sequence; or the heavy and light chain sequences of the antibodyare linked by a flexible linker such as consecutive glycine residues. Insome aspects, the immune modulator is a cytokine. In some aspects, thecytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21 orvariants thereof of each.

In some aspects, at least one of the at least one payload nucleic acidsequences are selected by performing the steps of: (a) obtaining atleast one of exome, transcriptome, or whole genome nucleotide sequencingdata from a tumor cell, an infected cell, or an infectious diseaseorganism, wherein the nucleotide sequencing data is used to obtain datarepresenting peptide sequences of each of a set of antigens; (b)inputting the peptide sequence of each antigen into a presentation modelto generate a set of numerical likelihoods that each of the antigens ispresented by one or more of the MHC alleles on a cell surface,optionally a tumor cell surface or an infected cell surface, the set ofnumerical likelihoods having been identified at least based on receivedmass spectrometry data; and (c) selecting a subset of the set ofantigens based on the set of numerical likelihoods to generate a set ofselected antigens which are used to generate the at least one payloadnucleic acid sequence.

In some aspects, each of the at least one payload nucleic acid sequencesare selected by performing the steps of: (a) obtaining at least one ofexome, transcriptome, or whole genome nucleotide sequencing data from atumor cell, an infected cell, or an infectious disease organism, whereinthe nucleotide sequencing data is used to obtain data representingpeptide sequences of each of a set of antigens; (b) inputting thepeptide sequence of each antigen into a presentation model to generate aset of numerical likelihoods that each of the antigens is presented byone or more of the MHC alleles on a cell surface, optionally a tumorcell surface or an infected cell surface, the set of numericallikelihoods having been identified at least based on received massspectrometry data; and (c) selecting a subset of the set of antigensbased on the set of numerical likelihoods to generate a set of selectedantigens which are used to generate each of the at least one payloadnucleic acid sequences. In some aspects, a number of the set of selectedantigens is 2-20. In some aspects, the presentation model representsdependence between: (a) presence of a pair of a particular one of theMHC alleles and a particular amino acid at a particular position of apeptide sequence; and (b) likelihood of presentation on a cell surface,optionally a tumor cell surface or an infected cell surface, by theparticular one of the MHC alleles of the pair, of such a peptidesequence comprising the particular amino acid at the particularposition. In some aspects, selecting the set of selected antigenscomprises selecting antigens that have an increased likelihood of beingpresented on the cell surface relative to unselected antigens based onthe presentation model. In some aspects, selecting the set of selectedantigens comprises selecting antigens that have an increased likelihoodof being capable of inducing a cell-specific immune response in thesubject relative to unselected antigens based on the presentation model.In some aspects, selecting the set of selected antigens comprisesselecting antigens that have an increased likelihood of being capable ofbeing presented to naïve T cells by professional antigen presentingcells (APCs) relative to unselected antigens based on the presentationmodel, optionally wherein the APC is a dendritic cell (DC). In someaspects, selecting the set of selected antigens comprises selectingantigens that have a decreased likelihood of being subject to inhibitionvia central or peripheral tolerance relative to unselected antigensbased on the presentation model. In some aspects, selecting the set ofselected antigens comprises selecting antigens that have a decreasedlikelihood of being capable of inducing an autoimmune response to normaltissue in the subject relative to unselected antigens based on thepresentation model. In some aspects, exome or transcriptome nucleotidesequencing data is obtained by performing sequencing on a tumor cell ortissue, an infected cell, or an infectious disease organism. In someaspects, the sequencing is next generation sequencing (NGS) or anymassively parallel sequencing approach.

In some aspects, the cassette comprises junctional epitope sequencesformed by adjacent sequences in the cassette. In some aspects, at leastone or each junctional epitope sequence has an affinity of greater than500 nM for MHC. In some aspects, each junctional epitope sequence isnon-self. In some aspects, the cassette does not encode anon-therapeutic MHC class I or class II epitope, wherein thenon-therapeutic epitope is predicted to be displayed on an MHC allele ofa subject. In some aspects, the non-therapeutic predicted MHC class I orclass II epitope sequence is a junctional epitope sequence formed byadjacent sequences in the cassette. In some aspects, the prediction inbased on presentation likelihoods generated by inputting sequences ofthe non-therapeutic epitopes into a presentation model. In some aspects,an order of the at least one payload nucleic acid sequences in thecassette is determined by a series of steps comprising: i. generating aset of candidate cassette sequences corresponding to different orders ofthe at least one payload nucleic acid sequences; ii. determining, foreach candidate cassette sequence, a presentation score based onpresentation of non-therapeutic epitopes in the candidate cassettesequence; and iii. selecting a candidate cassette sequence associatedwith a presentation score below a predetermined threshold as thecassette sequence.

In some aspects, each of the MHC class I and/or class II epitopes ispredicted or validated to be capable of presentation by at least one HLAallele present in at least 5% of a human population. In some aspects,each of the MHC class I and/or class II epitopes is predicted orvalidated to be capable of presentation by at least one HLA allele,wherein each antigen/HLA pair has an antigen/HLA prevalence of at least0.01% in a human population. In some aspects, each of the MHC class Iand/or class II epitopes is predicted or validated to be capable ofpresentation by at least one HLA allele, wherein each antigen/HLA pairhas an antigen/HLA prevalence of at least 0.1% in a human population. Insome aspects, the at least one payload nucleic acid sequence encodingthe polypeptide is codon optimized relative to a native nucleic acidsequence directly extracted from a subject tissue or sample.

Also disclosed herein is a pharmaceutical composition comprising any ofthe vectors described herein and a pharmaceutically acceptable carrier.In some aspects, the composition further comprises an adjuvant. In someaspects, the composition further comprises an immune modulator. In someaspects, the immune modulator is an anti-CTLA4 antibody or anantigen-binding fragment thereof, an anti-PD-1 antibody or anantigen-binding fragment thereof, an anti-PD-L1 antibody or anantigen-binding fragment thereof, an anti-4-1BB antibody or anantigen-binding fragment thereof, or an anti-OX-40 antibody or anantigen-binding fragment thereof.

Also disclosed herein is an isolated nucleotide sequence comprising thecassette of any of the vectors described herein and a gene of thesequence of SEQ ID NO: 1, optionally wherein the gene is selected fromthe group consisting of the chimpanzee adenovirus ITR, E1A, E1B, E2A,E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQID NO: 1, and optionally wherein the nucleotide sequence is cDNA.

Also disclosed herein is an isolated cell comprising any of the isolatednucleotide sequences described herein, optionally wherein the cell is aCHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, orAE1-2a cell.

Also disclosed is vector comprising any of the isolated nucleotidesequences described herein.

Also disclosed herein is a kit comprising any of the vectors orcompositions described herein and instructions for use.

Also disclosed herein is a method for stimulating an immune response ina subject, the method comprising administering to the subject any of thevectors or compositions described herein. In some aspects, the vector orcomposition is administered intramuscularly (IM), intradermally (ID), orsubcutaneously (SC). In some aspects, the method further comprisesadministering to the subject an immune modulator, optionally wherein theimmune modulator is administered before, concurrently with, or afteradministration of the vector or pharmaceutical composition. In someaspects, the immune modulator is an anti-CTLA4 antibody or anantigen-binding fragment thereof, an anti-PD-1 antibody or anantigen-binding fragment thereof, an anti-PD-L1 antibody or anantigen-binding fragment thereof, an anti-4-1BB antibody or anantigen-binding fragment thereof, or an anti-OX-40 antibody or anantigen-binding fragment thereof. In some aspects, the immune modulatoris administered intravenously (IV), intramuscularly (IM), intradermally(ID), or subcutaneously (SC). In some aspects, the subcutaneousadministration is near the site of the vector or compositionadministration or in close proximity to one or more vector orcomposition draining lymph nodes.

In some aspects, the method further comprises administering to thesubject a second vaccine composition. In some aspects, the secondvaccine composition is administered prior to the administration of anyof the vectors or compositions described herein. In some aspects, thesecond vaccine composition is administered subsequent to theadministration of any of the vectors or compositions described herein.In some aspects, the second vaccine composition is the same as any ofthe vectors or compositions described herein. In some aspects, thesecond vaccine composition is different from any of the vectors orcompositions described herein. In some aspects, the second vaccinecomposition comprises a self-amplifying RNA (samRNA) vector encoding atleast one payload nucleic acid sequence. In some aspects, the at leastone payload nucleic acid sequence encoded by the samRNA vector is thesame as at least one of the at least one payload nucleic acid sequenceof any of the above vector claims.

Also disclosed herein is a method of manufacturing the vector of any ofthe above vector claims, the method comprising: obtaining a plasmidsequence comprising the adenovirus vector or chimpanzee adenovirusvector; transfecting the plasmid sequence into one or more host cells;and isolating the vector from the one or more host cells. In someaspects, the isolating comprises: lysing the one or more host cells toobtain a cell lysate comprising the vector; and purifying the vectorfrom the cell lysate and optionally also from media used to culture theone or more host cells. In some aspects, the plasmid sequence isgenerated using one of the following; DNA recombination or bacterialrecombination or full genome DNA synthesis or full genome DNA synthesiswith amplification of synthesized DNA in bacterial cells. In someaspects, the one or more host cells are at least one of CHO, HEK293 orvariants thereof, 911, HeLa, A549, LP-293, PER.C6, and AE1-2a cells. Insome aspects, the purifying the vector from the cell lysate involves oneor more of chromatographic separation, centrifugation, virusprecipitation, and filtration.

Also provided herein is a method of producing a virus, wherein the virusis produced using any of the vectors described herein. In some aspects,the production of the virus is increased using the vector comprising thepartially deleted E4 gene relative to production of a virus producedusing a vector without the partially deleted E4 gene. In some aspects,the infectious unit titer of the virus is increased using the vectorcomprising the partially deleted E4 gene relative to the infectious unittiter of a virus produced using a vector without the partially deletedE4 gene. In some aspects, the increased production is increased at least1.5-fold, at least 2-fold, at least 2.5-fold, at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, or at least9-fold relative to production using a vector without the partiallydeleted E4 gene. In some aspects, the increased production is increasedat least 10-fold, at least 18-fold, at least 20-fold, at least 25-fold,or at least 27-fold, relative to production using a vector without thepartially deleted E4 gene.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 illustrates development of an in vitro T cell activation assay.Schematic of the assay in which the delivery of a vaccine cassette toantigen presenting cells, leads to expression, processing andMHC-restricted presentation of distinct peptide antigens. Reporter Tcells engineered with T cell receptors that match the specificpeptide-MHC combination become activated resulting in luciferaseexpression.

FIG. 2A illustrates evaluation of linker sequences in short cassettesand shows five class I MHC restricted epitopes (epitopes 1 through 5)concatenated in the same position relative to each other followed by twouniversal class II MHC epitopes (MHC-II). Various iterations weregenerated using different linkers. In some cases the T cell epitopes aredirectly linked to each other. In others, the T cell epitopes areflanked on one or both sides by its natural sequence. In otheriterations, the T cell epitopes are linked by the non-natural sequencesAAY, RR, and DPP.

FIG. 2B illustrates evaluation of linker sequences in short cassettesand shows sequence information on the T cell epitopes embedded in theshort cassettes. FIG. 2B discloses SEQ ID NOS 78-79, 82, 81, 80, 49 and194, respectively, in order of appearance.

FIG. 3 illustrates evaluation of cellular targeting sequences added tomodel vaccine cassettes. The targeting cassettes extend the shortcassette designs with ubiquitin (Ub), signal peptides (SP) and/ortransmembrane (TM) domains, feature next to the five marker human T cellepitopes (epitopes 1 through 5) also two mouse T cell epitopes SIINFEKL(SII) (SEQ ID NO: 72) and SPSYAYHQF (A5) (SEQ ID NO: 73), and use eitherthe non-natural linker AAY- or natural linkers flanking the T cellepitopes on both sides (25mer).

FIG. 4 illustrates in vivo evaluation of linker sequences in shortcassettes. A) Experimental design of the in vivo evaluation of vaccinecassettes using HLA-A2 transgenic mice.

FIG. 5A illustrates in vivo evaluation of the impact of epitope positionin long 21-mer cassettes and shows the design of long cassettes entailsfive marker class I epitopes (epitopes 1 through 5) contained in their25-mer natural sequence (linker=natural flanking sequences), spaced withadditional well-known T cell class I epitopes (epitopes 6 through 21)contained in their 25-mer natural sequence, and two universal class IIepitopes (MHC-110, with only the relative position of the class Iepitopes varied.

FIG. 5B illustrates in vivo evaluation of the impact of epitope positionin long 21-mer cassettes and shows the sequence information on the Tcell epitopes used. FIG. 5B discloses SEQ ID NOS 78-79, 82, 81, 80,195-197, 83 and 198-209, respectively, in order of appearance.

FIG. 6A illustrates final cassette design for preclinical IND-enablingstudies and shows the design of the final cassettes comprises 20 MHC Iepitopes contained in their 25-mer natural sequence (linker=naturalflanking sequences), composed of 6 non-human primate (NHP) epitopes, 5human epitopes, 9 murine epitopes, as well as 2 universal MHC class IIepitopes.

FIG. 6B illustrates final cassette design for preclinical IND-enablingstudies and shows the sequence information for the T cell epitopes usedthat are presented on class I MHC of non-human primate, mouse and humanorigin, as well as sequences of 2 universal MHC class II epitopes PADREand Tetanus toxoid. FIG. 6B discloses SEQ ID NOS 112-117, 80-82, 78-79,72-73, 142, 210, 146-148, 144-145, 49 and 47, respectively, in order ofcolumns.

FIG. 7A illustrates ChAdV68.4WTnt.GFP virus production aftertransfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNAusing the calcium phosphate protocol. Viral replication was observed 10days after transfection and ChAdV68.4WTnt.GFP viral plaques werevisualized using light microscopy (40× magnification).

FIG. 7B illustrates ChAdV68.4WTnt.GFP virus production aftertransfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNAusing the calcium phosphate protocol. Viral replication was observed 10days after transfection and ChAdV68.4WTnt.GFP viral plaques werevisualized using fluorescent microscopy at 40× magnification.

FIG. 7C illustrates ChAdV68.4WTnt.GFP virus production aftertransfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNAusing the calcium phosphate protocol. Viral replication was observed 10days after transfection and ChAdV68.4WTnt.GFP viral plaques werevisualized using fluorescent microscopy at 100× magnification.

FIG. 8A illustrates ChAdV68.5WTnt.GFP virus production aftertransfection. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNAusing the lipofectamine protocol. Viral replication (plaques) wasobserved 10 days after transfection. A lysate was made and used toreinfect a T25 flask of 293A cells. ChAdV68.5WTnt.GFP viral plaques werevisualized and photographed 3 days later using light microscopy (40×magnification)

FIG. 8B illustrates ChAdV68.5WTnt.GFP virus production aftertransfection. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNAusing the lipofectamine protocol. Viral replication (plaques) wasobserved 10 days after transfection. A lysate was made and used toreinfect a T25 flask of 293A cells. ChAdV68.5WTnt.GFP viral plaques werevisualized and photographed 3 days later using fluorescent microscopy at40× magnification.

FIG. 8C illustrates ChAdV68.5WTnt.GFP virus production aftertransfection. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNAusing the lipofectamine protocol. Viral replication (plaques) wasobserved 10 days after transfection. A lysate was made and used toreinfect a T25 flask of 293A cells. ChAdV68.5WTnt.GFP viral plaques werevisualized and photographed 3 days later using fluorescent microscopy at100× magnification.

FIG. 9 illustrates the viral particle production scheme.

FIG. 10 illustrates the alphavirus derived VEE self-replicating RNA(srRNA) vector.

FIG. 11 illustrates in vivo reporter expression after inoculation ofC57BL/6J mice with VEE-Luciferase srRNA. Shown are representative imagesof luciferase signal following immunization of C57BL/6J mice withVEE-Luciferase srRNA (10 ug per mouse, bilateral intramuscularinjection, MC3 encapsulated) at various timepoints.

FIG. 12A illustrates T-cell responses measured 14 days afterimmunization with VEE srRNA formulated with MC3 LNP in B16-OVA tumorbearing mice. B16-OVA tumor bearing C57BL/6J mice were injected with 10ug of VEE-Luciferase srRNA (control), VEE-UbAAY srRNA (Vax),VEE-Luciferase srRNA and anti-CTLA-4 (aCTLA-4) or VEE-UbAAY srRNA andanti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated withanti-PD1 mAb starting at day 7. Each group consisted of 8 mice. Micewere sacrificed and spleens and lymph nodes were collected 14 days afterimmunization. SIINFEKL-specific T-cell (“SIINFEKL” disclosed as SEQ IDNO: 72) responses were assessed by IFN-gamma ELISPOT and are reported asspot-forming cells (SFC) per 106 splenocytes. Lines represent medians.

FIG. 12B illustrates T-cell responses measured 14 days afterimmunization with VEE srRNA formulated with MC3 LNP in B16-OVA tumorbearing mice. B16-OVA tumor bearing C57BL/6J mice were injected with 10ug of VEE-Luciferase srRNA (control), VEE-UbAAY srRNA (Vax),VEE-Luciferase srRNA and anti-CTLA-4 (aCTLA-4) or VEE-UbAAY srRNA andanti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated withanti-PD1 mAb starting at day 7. Each group consisted of 8 mice. Micewere sacrificed and spleens and lymph nodes were collected 14 days afterimmunization. SIINFEKL-specific T-cell (“SIINFEKL” disclosed as SEQ IDNO: 72) responses were assessed by MHCI-pentamer staining, reported aspentamer positive cells as a percent of CD8 positive cells. Linesrepresent medians.

FIG. 13A illustrates antigen-specific T-cell responses followingheterologous prime/boost in B16-OVA tumor bearing mice. B16-OVA tumorbearing C57BL/6J mice were injected with adenovirus expressing GFP(Ad5-GFP) and boosted with VEE-Luciferase srRNA formulated with MC3 LNP(Control) or Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both theControl and Vax groups were also treated with an IgG control mAb. Athird group was treated with the Ad5-GFP prime/VEE-Luciferase srRNAboost in combination with anti-CTLA-4 (aCTLA-4), while the fourth groupwas treated with the Ad5-UbAAY prime/VEE-UbAAY boost in combination withanti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated withanti-PD-1 mAb starting at day 21. T-cell responses were measured byIFN-gamma ELISPOT. Mice were sacrificed and spleens and lymph nodescollected at 14 days post immunization with adenovirus.

FIG. 13B illustrates antigen-specific T-cell responses followingheterologous prime/boost in B16-OVA tumor bearing mice. B16-OVA tumorbearing C57BL/6J mice were injected with adenovirus expressing GFP(Ad5-GFP) and boosted with VEE-Luciferase srRNA formulated with MC3 LNP(Control) or Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both theControl and Vax groups were also treated with an IgG control mAb. Athird group was treated with the Ad5-GFP prime/VEE-Luciferase srRNAboost in combination with anti-CTLA-4 (aCTLA-4), while the fourth groupwas treated with the Ad5-UbAAY prime/VEE-UbAAY boost in combination withanti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated withanti-PD-1 mAb starting at day 21. T-cell responses were measured byIFN-gamma ELISPOT. Mice were sacrificed and spleens and lymph nodescollected at 14 days post immunization with adenovirus and 14 days postboost with srRNA (day 28 after prime).

FIG. 13C illustrates antigen-specific T-cell responses followingheterologous prime/boost in B16-OVA tumor bearing mice. B16-OVA tumorbearing C57BL/6J mice were injected with adenovirus expressing GFP(Ad5-GFP) and boosted with VEE-Luciferase srRNA formulated with MC3 LNP(Control) or Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both theControl and Vax groups were also treated with an IgG control mAb. Athird group was treated with the Ad5-GFP prime/VEE-Luciferase srRNAboost in combination with anti-CTLA-4 (aCTLA-4), while the fourth groupwas treated with the Ad5-UbAAY prime/VEE-UbAAY boost in combination withanti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated withanti-PD-1 mAb starting at day 21. T-cell responses were measured by MHCclass I pentamer staining. Mice were sacrificed and spleens and lymphnodes collected at 14 days post immunization with adenovirus.

FIG. 13D illustrates antigen-specific T-cell responses followingheterologous prime/boost in B16-OVA tumor bearing mice. B16-OVA tumorbearing C57BL/6J mice were injected with adenovirus expressing GFP(Ad5-GFP) and boosted with VEE-Luciferase srRNA formulated with MC3 LNP(Control) or Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both theControl and Vax groups were also treated with an IgG control mAb. Athird group was treated with the Ad5-GFP prime/VEE-Luciferase srRNAboost in combination with anti-CTLA-4 (aCTLA-4), while the fourth groupwas treated with the Ad5-UbAAY prime/VEE-UbAAY boost in combination withanti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated withanti-PD-1 mAb starting at day 21. T-cell responses were measured by MHCclass I pentamer staining. Mice were sacrificed and spleens and lymphnodes collected at 14 days post immunization with adenovirus and 14 dayspost boost with srRNA (day 28 after prime).

FIG. 14A illustrates antigen-specific T-cell responses followingheterologous prime/boost in CT26 (Balb/c) tumor bearing mice. Mice wereimmunized with Ad5-GFP and boosted 15 days after the adenovirus primewith VEE-Luciferase srRNA formulated with MC3 LNP (Control) or primedwith Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the Controland Vax groups were also treated with an IgG control mAb. A separategroup was administered the Ad5-GFP/VEE-Luciferase srRNA prime/boost incombination with anti-PD-1 (aPD1), while a fourth group received theAd5-UbAAY/VEE-UbAAY srRNA prime/boost in combination with an anti-PD-1mAb (Vax+aPD1). T-cell responses to the AH1 peptide were measured usingIFN-gamma ELISPOT. Mice were sacrificed and spleens and lymph nodescollected at 12 days post immunization with adenovirus.

FIG. 14B illustrates antigen-specific T-cell responses followingheterologous prime/boost in CT26 (Balb/c) tumor bearing mice. Mice wereimmunized with Ad5-GFP and boosted 15 days after the adenovirus primewith VEE-Luciferase srRNA formulated with MC3 LNP (Control) or primedwith Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the Controland Vax groups were also treated with an IgG control mAb. A separategroup was administered the Ad5-GFP/VEE-Luciferase srRNA prime/boost incombination with anti-PD-1 (aPD1), while a fourth group received theAd5-UbAAY/VEE-UbAAY srRNA prime/boost in combination with an anti-PD-1mAb (Vax+aPD1). T-cell responses to the AH1 peptide were measured usingIFN-gamma ELISPOT. Mice were sacrificed and spleens and lymph nodescollected at 12 days post immunization with adenovirus and 6 days postboost with srRNA (day 21 after prime).

FIG. 15 illustrates ChAdV68 eliciting T-Cell responses to mouse tumorantigens in mice. Mice were immunized with ChAdV68.5WTnt.MAG25mer, andT-cell responses to the MHC class I epitope SIINFEKL (SEQ ID NO: 72)(OVA) were measured in C57BL/6J female mice and the MHC class I epitopeAH1-A5 measured in Balb/c mice. Mean spot forming cells (SFCs) per 10⁶splenocytes measured in ELISpot assays presented. Error bars representstandard deviation.

FIG. 16 illustrates cellular immune responses in a CT26 tumor modelfollowing a single immunization with either ChAdV6, ChAdV+anti-PD-1,srRNA, srRNA+anti-PD-1, or anti-PD-1 alone. Antigen-specific IFN-gammaproduction was measured in splenocytes for 6 mice from each group usingELISpot. Results are presented as spot forming cells (SFC) per 10⁶splenocytes. Median for each group indicated by horizontal line. Pvalues determined using the Dunnett's multiple comparison test; ***P<0.0001, **P<0.001, *P<0.05. ChAdV=ChAdV68.5WTnt.MAG25mer;srRNA=VEE-MAG25mer srRNA.

FIG. 17 illustrates CD8 T-Cell responses in a CT26 tumor model followinga single immunization with either ChAdV6, ChAdV+anti-PD-1, srRNA,srRNA+anti-PD-1, or anti-PD-1 alone. Antigen-specific IFN-gammaproduction in CD8 T cells measured using ICS and results presented asantigen-specific CD8 T cells as a percentage of total CD8 T cells.Median for each group indicated by horizontal line. P values determinedusing the Dunnett's multiple comparison test; *** P<0.0001, **P<0.001,*P<0.05. ChAdV=ChAdV68.5WTnt.MAG25mer; srRNA=VEE-MAG25mer srRNA.

FIG. 18 illustrates tumor growth in a CT26 tumor model followingimmunization with a ChAdV/srRNA heterologous prime/boost, a srRNA/ChAdVheterologous prime/boost, or a srRNA/srRNA homologous primer/boost. Alsoillustrated in a comparison of the prime/boost immunizations with orwithout administration of anti-PD1 during prime and boost. Tumor volumesmeasured twice per week and mean tumor volumes presented for the first21 days of the study. 22-28 mice per group at study initiation. Errorbars represent standard error of the mean (SEM). P values determinedusing the Dunnett's test; *** P<0.0001, **P<0.001, *P<0.05.ChAdV=ChAdV68.5WTnt.MAG25mer; srRNA=VEE-MAG25mer srRNA.

FIG. 19 illustrates survival in a CT26 tumor model followingimmunization with a ChAdV/srRNA heterologous prime/boost, a srRNA/ChAdVheterologous prime/boost, or a srRNA/srRNA homologous primer/boost. Alsoillustrated in a comparison of the prime/boost immunizations with orwithout administration of anti-PD1 during prime and boost. P valuesdetermined using the log-rank test; *** P<0.0001, **P<0.001, *P<0.01.ChAdV=ChAdV68.5WTnt.MAG25mer; srRNA=VEE-MAG25mer srRNA.

FIG. 20A illustrates antigen-specific cellular immune responses measuredusing ELISpot. Antigen-specific IFN-gamma production to six differentmamu A01 restricted epitopes was measured in PBMCs for the VEE-MAG25mersrRNA-LNP1(30 μg) using ELISpot 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeksafter the initial immunization (6 rhesus macaques per group). Resultsare presented as mean spot forming cells (SFC) per 10⁶ PBMCs for eachepitope in a stacked bar graph format. Values for each animal werenormalized to the levels at pre-bleed (week 0).

FIG. 20B illustrates antigen-specific cellular immune responses measuredusing ELISpot. Antigen-specific IFN-gamma production to six differentmamu A01 restricted epitopes was measured in PBMCs for the VEE-MAG25mersrRNA-LNP1(100 μg) using ELISpot 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeksafter the initial immunization (6 rhesus macaques per group). Resultsare presented as mean spot forming cells (SFC) per 10⁶ PBMCs for eachepitope in a stacked bar graph format. Values for each animal werenormalized to the levels at pre-bleed (week 0)

FIG. 20C illustrates antigen-specific cellular immune responses measuredusing ELISpot. Antigen-specific IFN-gamma production to six differentmamu A01 restricted epitopes was measured in PBMCs for the VEE-MAG25mersrRNA-LNP2(100 μg) homologous prime/boost using ELISpot 1, 2, 3, 4, 5,6, 8, 9, or 10 weeks after the initial immunization (6 rhesus macaquesper group). Results are presented as mean spot forming cells (SFC) per10⁶ PBMCs for each epitope in a stacked bar graph format. Values foreach animal were normalized to the levels at pre-bleed (week 0).

FIG. 20D illustrates antigen-specific cellular immune responses measuredusing ELISpot. Antigen-specific IFN-gamma production to six differentmamu A01 restricted epitopes was measured in PBMCs for theChAdV68.5WTnt.MAG25mer/VEE-MAG25mer srRNA heterologous prime/boost groupusing ELISpot 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after the initialimmunization (6 rhesus macaques per group). Results are presented asmean spot forming cells (SFC) per 10⁶ PBMCs for each epitope in astacked bar graph format. Values for each animal were normalized to thelevels at pre-bleed (week 0).

FIG. 21 shows antigen-specific cellular immune response measured usingELISpot. Antigen-specific IFN-gamma production to six different mamu A01restricted epitopes was measured in PBMCs after immunization with theChAdV68.5WTnt.MAG25mer/VEE-MAG25mer srRNA heterologous prime/boostregimen using ELISpot prior to immunization and 4, 5, 6, 7, 8, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks after theinitial immunization. Results are presented as mean spot forming cells(SFC) per 10⁶ PBMCs for each epitope (6 rhesus macaques per group) in astacked bar graph format.

FIG. 22 shows antigen-specific cellular immune response measured usingELISpot. Antigen-specific IFN-gamma production to six different mamu A01restricted epitopes was measured in PBMCs after immunization with theVEE-MAG25mer srRNA LNP2 homologous prime/boost regimen using ELISpotprior to immunization and 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeksafter the initial immunization. Results are presented as mean spotforming cells (SFC) per 10⁶ PBMCs for each epitope (6 rhesus macaquesper group) in a stacked bar graph format.

FIG. 23 shows antigen-specific cellular immune response measured usingELISpot. Antigen-specific IFN-gamma production to six different mamu A01restricted epitopes was measured in PBMCs after immunization with theVEE-MAG25mer srRNA LNP1 homologous prime/boost regimen using ELISpotprior to immunization and 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeksafter the initial immunization. Results are presented as mean spotforming cells (SFC) per 10⁶ PBMCs for each epitope (6 rhesus macaquesper group) in a stacked bar graph format.

FIG. 24A shows an example peptide spectrum generated from Promega'sdynamic range standard. FIG. 24A discloses SEQ ID NO: 77.

FIG. 24B shows an example peptide spectrum generated from Promega'sdynamic range standard.

FIG. 25 shows productivity, as assessed by IU titers, of the eightselected ChAdV68-MAG rapidly growing plaques compared to thenon-purified pooled virus. Numbers above the columns on the graphindicate fold improvement over the pooled virus in a controlledinfection at an MOI of 0.1.

FIG. 26 shows a schematic of the E4 locus and the 727 bp deletionbetween E4orf2-E4orf4 identified in Clone 1A.

FIG. 27 shows virus productivity with viruses plus and minus the E4deletion. Numbers above the bar indicate fold improvement over non-E4deleted virus. The ChAdV68-MAG comparison to ChAdV68-MAG-E4 virus wasperformed on 3 separate occasions. In each case a 400 mL production runwith both viruses was performed at an MOI of 1.0. Shown are viralparticle (VP) titers (left panel) and infectious unit (IU) titers (rightpanel).

FIG. 28 shows a Western blot analysis of MAG expression using rabbitanti-class II epitope antibody expression in cells infected withChAdV68.5WTnt.MAG25mer (“MAG”) and ChAdV68-MAG-E4deleted (“MAG-E4”)viruses. Samples were treated with and without the proteasome inhibitor,MG-132, as indicated by plus and minus signs.

FIG. 29 illustrates the general organization of the model epitopes fromthe various species for large antigen cassettes that had either 30 (L),40 (XL) or 50 (XXL) epitopes.

FIG. 30 shows ChAd vectors express long cassettes as indicated by theabove Western blot using an anti-class II (PADRE) antibody thatrecognizes a sequence common to all cassettes. HEK293 cells wereinfected with ChAdV68 vectors expressing large cassettes (ChAdV68-50XXL,ChAdV68-40XL & ChAdV68-30L) of variable size. Infections were set up ata MOI of 0.2. Twenty-four hours post infection MG132 a proteasomeinhibitor was added to a set of the infected wells (indicated by theplus sign). Another set of virus treated wells were not treated withMG132 (indicated by minus sign). Uninfected HEK293 cells (293F) wereused as a negative control. Forty-eight hours post infection cellpellets were harvested and analyzed by SDS/PAGE electrophoresis, andimmunoblotting using a rabbit anti-Class II PADRE antibody. A HRPanti-rabbit antibody and ECL chemiluminescent substrate was used fordetection.

FIG. 31 shows CD8+ immune responses in ChAdV68 large cassette immunizedmice, detected against AH1 (top) and SIINFEKL (SEQ ID NO: 72) (bottom)by ICS. Data is presented as IFNg+ cells against the model epitope as %of total CD8 cells

FIG. 32 shows CD8+ responses to LD-AH1+(top) and Kb-SIINFEKL+ (bottom)(“SIINFEKL” disclosed as SEQ ID NO: 72) Tetramers post ChAdV68 largecassette vaccination. Data is presented as % of total CD8 cells reactiveagainst the model Tetramer peptide complex. *p<0.05, **p<0.01 by ANOVAwith Tukey's test. All p-values compared to MAG 20-antigen cassette.

FIG. 33 shows CD8+ immune responses in alphavirus large cassette treatedmice, detected against AH1 (top) and SIINFEKL (SEQ ID NO: 72) (bottom)by ICS. Data is presented as IFNg+ cells against the model epitope as %of total CD8 cells. *p<0.05, **p<0.01, ***p<0.001 by ANOVA with Tukey'stest. All p-values compared to MAG 20-antigen cassette.

FIG. 34 illustrates the vaccination strategy used to evaluateimmunogenicity of the antigen-cassette containing vectors in rhesusmacaques. Triangles indicate ChAdV68 vaccination (1e12 vp/animal) atweeks 0 & 32. Circles represent alphavirus vaccination at weeks 0, 4,12, 20, 28 & 32. Squares represent administration of an anti-CTLA4antibody.

FIG. 35 shows a time course of CD8+ anti-epitope responses in RhesusMacaques dosed with chAd-MAG alone (Group 4). Mean SFC/1e6 splenocytesis shown.

FIG. 36 shows a time course of CD8+ anti-epitope responses in RhesusMacaques dosed with chAd-MAG plus anti-CTLA4 antibody (Ipilimumab)delivered IV (Group 5). Mean SFC/1e6 splenocytes is shown.

FIG. 37 shows a time course of CD8+ anti-epitope responses in RhesusMacaques dosed with chAd-MAG plus anti-CTLA4 antibody (Ipilimumab)delivered SC (Group 6). Mean SFC/1e6 splenocytes is shown.

FIG. 38 shows antigen-specific memory responses generated byChAdV68/samRNA vaccine protocol measured by ELISpot. Results arepresented as individual dot plots, with each dot representing a singleanimal. Pre-immunization baseline (left panel) and memory response at 18months post-prime (right panel) are shown.

FIG. 39 shows memory cell phenotyping of antigen-specific CD8+ T-cellsby flow cytometry using combinatorial tetramer staining and CD45RA/CCR7co-staining.

FIG. 40 shows the distribution of memory cell types within the sum ofthe four Mamu-A*01 tetramer+ CD8+ T-cell populations at study month 18.Memory cells were characterized as follows: CD45RA+CCR7+=naïve,CD45RA+CCR7-=effector (Teff), CD45RA-CCR7+=central memory (Tcm),CD45RA-CCR7-=effector memory (Tem).

FIG. 41 shows frequency of CD8+ T cells recognizing the CT26 tumorantigen AH1 in CT26 tumor-bearing mice. P values determined using theone-way ANOVA with Tukey's multiple comparisons test; **P<0.001,*P<0.05. ChAdV=ChAdV68.5WTnt.MAG25mer; aCTLA4=anti-CTLA4 antibody, clone9D9.

FIG. 42A shows the CD8+ immune responses by assessing IFN-gammaproduction by ICS following stimulation with an AH1 (a dominant epitopefrom Murine leukemia virus envelope protein gp70) in ChAdV68-MAG andChAdV68-E4delta-MAG vector treated Balb/c mice. Balb/c mice wereimmunized by bilateral injection of 50 uL of virus into the Quadriceps(100 uL in total, 50 uL/leg).

FIG. 42B shows T cell responses by assessing IFN-gamma production byELISpot following stimulation with 6 different rhesus macaque Mamu-A*01class I epitopes at week 2 in Rhesus macaques were immunized withChAdV68-CMV-MAG (left panel) and ChAdV68-E4d-CMT-MAG (right panel), andboth conditions administered an anti-CTLA4 antibody (Ipilimumab).

FIG. 42C shows T cell responses by assessing IFN-gamma production byELISpot following stimulation with 6 different rhesus macaque Mamu-A*01class I epitopes over a time course in Rhesus macaques were immunizedwith ChAdV68-CMV-MAG (left panel) and ChAdV68-E4d-CMT-MAG (right panel),and both conditions administered an anti-CTLA4 antibody (Ipilimumab).

FIG. 43 illustrates the general strategy for a tetracycline-controlledviral production system using the example of antigen encoding vaccine.

FIG. 44A presents a schematic showing arrangement of a “TETo” responseregion in reference to the promoter and cassette to be expressed.

FIG. 44B presents a schematic showing arrangement of a “CMT” responseregion in reference to the promoter and cassette to be expressed.

FIG. 45A shows TETr mediated regulation of GFP expressed from a ChAdV68vector with a TETo sequence. GFP is significantly reduced in 293F cellsexpressing the TETr (Clone 17, right panel) relative to the parental293F cell line (left panel). Cells were infected at an MOI of 1 withChAdV68-TETo-GFP and 24 h later GFP was evaluated by florescentmicroscopy under a 10× objective.

FIG. 45B shows TETr mediated regulation of SEAP expressed from a ChAdV68vector with a CMT sequence. SEAP is significantly reduced in 293F cellsexpressing the TETr (Clone 17, second column from left) relative to theparental 293F cell line (left column). Background signal was establishedusing a ChAdV68 vector expressing a control expression cassette (righttwo columns). 293F cells were infected at an MOI of 0.3 and 24 h latermedia was harvested for the SEAP assay (Phospha-Light™ System (AppliedBiosystems) using a chemiluminescent substrate for the detection ofsecreted alkaline phosphatase) that was followed according to themanufacturers description.

FIG. 46 shows viral production for a ChAdV68-Teto-MAG vector in a 293FTETr repressor line (Clone 17) relative to production in the parental293F line. The experiment was performed in triplicate (run 1-3). In eachexperiment 400 mL of 293F cells were infected at an MOI of approximately3 and incubated for 48-72 h before harvesting. Virus was purified by twodiscontinuous CsCl ultracentrifugation steps and dialyzed into storagebuffer. Viral particles were measured by Absorbance at 260 nm. Shown areviral particle (VP; top panels) and infectious unit (IU; bottom panels)titers.

FIG. 47A shows overall productivity of a Tet regulated virus(“TETo-MAG”) in a 293F TETr line (Clone 17) relative to a non-regulatedvirus (“MAG”) with the same cassette in a normal 293F cell line. Shownare date from multiple 400 mL production runs followed bycentrifugation. Fold improvement with Tet regulated virus is indicatedby the number above the graph.

FIG. 47B shows viral production for the ChAdV68-CT-TSNA,ChAdV68-TETo-TSNA, ChAdV68-CMT-TSNA, and ChAdV68-E4d-CMT-TSNA virusesrelative to ChAdV68-CMV-TSNA.

FIG. 47C shows viral production for model antigen cassettes 50XXL andM2.2 using adenoviral vectors having a CMT response region in a tTSexpressing cell line.

FIG. 48 shows antigen specific T-Cell responses following vaccinationwith regulated versus no-regulated vectors. Antigen-specific IFN-gammaproduction in CD8 T cells measured using ICS and results presented asantigen-specific CD8 T cells as a percentage of total CD8 T cells.Median for each group indicated by horizontal line. Balb/c mice wereimmunized with 1×10¹⁰ VP of ChAdV68 vaccines expressing a model antigencassette either under control of normal CMV promoter (ChAdV-MAG) or aTETo regulated promoter (TET-ChAdV-MAG). 12 d post vaccination spleenswere harvested and single cell suspensions made.

DETAILED DESCRIPTION I. Definitions

In general, terms used in the claims and the specification are intendedto be construed as having the plain meaning understood by a person ofordinary skill in the art. Certain terms are defined below to provideadditional clarity. In case of conflict between the plain meaning andthe provided definitions, the provided definitions are to be used.

As used herein the term “antigen” is a substance that induces an immuneresponse. An antigen can be a neoantigen. An antigen can be a “sharedantigen” that is an antigen found among a specific population, e.g., aspecific population of cancer patients or infected subjects. An antigencan be associated with or derived from an infectious disease organism.

As used herein the term “neoantigen” is an antigen that has at least onealteration that makes it distinct from the corresponding wild-typeantigen, e.g., via mutation in a tumor cell or post-translationalmodification specific to a tumor cell. A neoantigen can include apolypeptide sequence or a nucleic acid sequence. A mutation can includea frameshift or nonframeshift indel, missense or nonsense substitution,splice site alteration, genomic rearrangement or gene fusion, or anygenomic or expression alteration giving rise to a neoORF. A mutationscan also include a splice variant. Post-translational modificationsspecific to a tumor cell can include aberrant phosphorylation.Post-translational modifications specific to a tumor cell can alsoinclude a proteasome-generated spliced antigen. See Liepe et al., Alarge fraction of HLA class I ligands are proteasome-generated splicedpeptides; Science. 2016 Oct. 21; 354(6310):354-358. The subject can beidentified for administration through the use of various diagnosticmethods, e.g., patient selection methods described further below.

As used herein the term “tumor antigen” is an antigen present in asubject's tumor cell or tissue but not in the subject's correspondingnormal cell or tissue, or derived from a polypeptide known to or havebeen found to have altered expression in a tumor cell or canceroustissue in comparison to a normal cell or tissue.

As used herein the term “antigen-based vaccine” is a vaccine compositionbased on one or more antigens, e.g., a plurality of antigens. Thevaccines can be nucleotide-based (e.g., virally based, RNA based, or DNAbased), protein-based (e.g., peptide based), or a combination thereof.

As used herein the term “candidate antigen” is a mutation or otheraberration giving rise to a sequence that may represent an antigen.

As used herein the term “coding region” is the portion(s) of a gene thatencode protein.

As used herein the term “coding mutation” is a mutation occurring in acoding region.

As used herein the term “ORF” means open reading frame.

As used herein the term “NEO-ORF” is a tumor-specific ORF arising from amutation or other aberration such as splicing.

As used herein the term “missense mutation” is a mutation causing asubstitution from one amino acid to another.

As used herein the term “nonsense mutation” is a mutation causing asubstitution from an amino acid to a stop codon or causing removal of acanonical start codon.

As used herein the term “frameshift mutation” is a mutation causing achange in the frame of the protein.

As used herein the term “indel” is an insertion or deletion of one ormore nucleic acids.

As used herein, the term percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms described below (e.g., BLASTP and BLASTNor other algorithms available to persons of skill) or by visualinspection. Depending on the application, the percent “identity” canexist over a region of the sequence being compared, e.g., over afunctional domain, or, alternatively, exist over the full length of thetwo sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Alternatively, sequence similarity ordissimilarity can be established by the combined presence or absence ofparticular nucleotides, or, for translated sequences, amino acids atselected sequence positions (e.g., sequence motifs).

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al.).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information.

As used herein the term “non-stop or read-through” is a mutation causingthe removal of the natural stop codon.

As used herein the term “epitope” is the specific portion of an antigentypically bound by an antibody or T cell receptor.

As used herein the term “immunogenic” is the ability to elicit an immuneresponse, e.g., via T cells, B cells, or both.

As used herein the term “HLA binding affinity” “MHC binding affinity”means affinity of binding between a specific antigen and a specific MHCallele.

As used herein the term “bait” is a nucleic acid probe used to enrich aspecific sequence of DNA or RNA from a sample.

As used herein the term “variant” is a difference between a subject'snucleic acids and the reference human genome used as a control.

As used herein the term “variant call” is an algorithmic determinationof the presence of a variant, typically from sequencing.

As used herein the term “polymorphism” is a germline variant, i.e., avariant found in all DNA-bearing cells of an individual.

As used herein the term “somatic variant” is a variant arising innon-germline cells of an individual.

As used herein the term “allele” is a version of a gene or a version ofa genetic sequence or a version of a protein.

As used herein the term “HLA type” is the complement of HLA genealleles.

As used herein the term “nonsense-mediated decay” or “NMD” is adegradation of an mRNA by a cell due to a premature stop codon.

As used herein the term “truncal mutation” is a mutation originatingearly in the development of a tumor and present in a substantial portionof the tumor's cells.

As used herein the term “subclonal mutation” is a mutation originatinglater in the development of a tumor and present in only a subset of thetumor's cells.

As used herein the term “exome” is a subset of the genome that codes forproteins. An exome can be the collective exons of a genome.

As used herein the term “logistic regression” is a regression model forbinary data from statistics where the logit of the probability that thedependent variable is equal to one is modeled as a linear function ofthe dependent variables.

As used herein the term “neural network” is a machine learning model forclassification or regression consisting of multiple layers of lineartransformations followed by element-wise nonlinearities typicallytrained via stochastic gradient descent and back-propagation.

As used herein the term “proteome” is the set of all proteins expressedand/or translated by a cell, group of cells, or individual.

As used herein the term “peptidome” is the set of all peptides presentedby MHC-I or MHC-II on the cell surface. The peptidome may refer to aproperty of a cell or a collection of cells (e.g., the tumor peptidome,meaning the union of the peptidomes of all cells that comprise thetumor).

As used herein the term “ELISPOT” means Enzyme-linked immunosorbent spotassay—which is a common method for monitoring immune responses in humansand animals.

As used herein the term “dextramers” is a dextran-based peptide-MHCmultimers used for antigen-specific T-cell staining in flow cytometry.

As used herein the term “tolerance or immune tolerance” is a state ofimmune non-responsiveness to one or more antigens, e.g. self-antigens.

As used herein the term “central tolerance” is a tolerance affected inthe thymus, either by deleting self-reactive T-cell clones or bypromoting self-reactive T-cell clones to differentiate intoimmunosuppressive regulatory T-cells (Tregs).

As used herein the term “peripheral tolerance” is a tolerance affectedin the periphery by downregulating or anergizing self-reactive T-cellsthat survive central tolerance or promoting these T cells todifferentiate into Tregs.

The term “sample” can include a single cell or multiple cells orfragments of cells or an aliquot of body fluid, taken from a subject, bymeans including venipuncture, excretion, ejaculation, massage, biopsy,needle aspirate, lavage sample, scraping, surgical incision, orintervention or other means known in the art.

The term “subject” encompasses a cell, tissue, or organism, human ornon-human, whether in vivo, ex vivo, or in vitro, male or female. Theterm subject is inclusive of mammals including humans.

The term “mammal” encompasses both humans and non-humans and includesbut is not limited to humans, non-human primates, canines, felines,murines, bovines, equines, and porcines.

The term “clinical factor” refers to a measure of a condition of asubject, e.g., disease activity or severity. “Clinical factor”encompasses all markers of a subject's health status, includingnon-sample markers, and/or other characteristics of a subject, such as,without limitation, age and gender. A clinical factor can be a score, avalue, or a set of values that can be obtained from evaluation of asample (or population of samples) from a subject or a subject under adetermined condition. A clinical factor can also be predicted by markersand/or other parameters such as gene expression surrogates. Clinicalfactors can include tumor type, tumor sub-type, and smoking history.

The term “derived” refers to sequences directly extracted from a subjecttissue or sample (e.g., a tumor, cell, infected cell, or infectiousdisease organism), e.g. via RT-PCR; or sequence data obtained bysequencing the subject tissue or sample and then synthesizing thenucleic acid sequences using the sequencing data, e.g., via varioussynthetic or PCR-based methods known in the art. “Derived” can includenucleic acid sequence variants, such as codon-optimized nucleic acidsequence variants, that encode the same polypeptide sequence as acorresponding native nucleic acid sequence, such as a correspondingnative infectious disease organism nucleic acid sequence. “Derived” canalso include variants that encode a modified polypeptide sequence, suchas an infectious disease organism polypeptide sequence, having one ormore (e.g., 1, 2, 3, 4, or 5) mutations relative to a native polypeptidesequence, such as native infectious disease organism polypeptidesequence. For example, a modified polypeptide sequence can have one ormore missense mutations (e.g., engineered mutations) relative to thenative polypeptide sequence.

The term “alphavirus” refers to members of the family Togaviridae, andare positive-sense single-stranded RNA viruses. Alphaviruses aretypically classified as either Old World, such as Sindbis, Ross River,Mayaro, Chikungunya, and Semliki Forest viruses, or New World, such aseastern equine encephalitis, Aura, Fort Morgan, or Venezuelan equineencephalitis and its derivative strain TC-83. Alphaviruses are typicallyself-replicating RNA viruses.

The term “alphavirus backbone” refers to minimal sequence(s) of analphavirus that allow for self-replication of the viral genome. Minimalsequences can include conserved sequences for nonstructuralprotein-mediated amplification, a nonstructural protein 1 (nsP1) gene, ansP2 gene, a nsP3 gene, a nsP4 gene, and a polyA sequence, as well assequences for expression of subgenomic viral RNA including a 26Spromoter element.

The term “sequences for nonstructural protein-mediated amplification”includes alphavirus conserved sequence elements (CSE) well known tothose in the art. CSEs include, but are not limited to, an alphavirus 5′UTR, a 51-nt CSE, a 24-nt CSE, or other 26S subgenomic promotersequence, a 19-nt CSE, and an alphavirus 3′ UTR.

The term “RNA polymerase” includes polymerases that catalyze theproduction of RNA polynucleotides from a DNA template. RNA polymerasesinclude, but are not limited to, bacteriophage derived polymerasesincluding T3, T7, and SP6.

The term “lipid” includes hydrophobic and/or amphiphilic molecules.Lipids can be cationic, anionic, or neutral. Lipids can be synthetic ornaturally derived, and in some instances biodegradable. Lipids caninclude cholesterol, phospholipids, lipid conjugates including, but notlimited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids),waxes, oils, glycerides, fats, and fat-soluble vitamins. Lipids can alsoinclude dilinoleylmethyl-4-dimethylaminobutyrate (MC3) and MC3-likemolecules.

The term “lipid nanoparticle” or “LNP” includes vesicle like structuresformed using a lipid containing membrane surrounding an aqueousinterior, also referred to as liposomes. Lipid nanoparticles includeslipid-based compositions with a solid lipid core stabilized by asurfactant. The core lipids can be fatty acids, acylglycerols, waxes,and mixtures of these surfactants. Biological membrane lipids such asphospholipids, sphingomyelins, bile salts (sodium taurocholate), andsterols (cholesterol) can be utilized as stabilizers. Lipidnanoparticles can be formed using defined ratios of different lipidmolecules, including, but not limited to, defined ratios of one or morecationic, anionic, or neutral lipids. Lipid nanoparticles canencapsulate molecules within an outer-membrane shell and subsequentlycan be contacted with target cells to deliver the encapsulated moleculesto the host cell cytosol. Lipid nanoparticles can be modified orfunctionalized with non-lipid molecules, including on their surface.Lipid nanoparticles can be single-layered (unilamellar) or multi-layered(multilamellar). Lipid nanoparticles can be complexed with nucleic acid.Unilamellar lipid nanoparticles can be complexed with nucleic acid,wherein the nucleic acid is in the aqueous interior. Multilamellar lipidnanoparticles can be complexed with nucleic acid, wherein the nucleicacid is in the aqueous interior, or to form or sandwiched between

Abbreviations: MHC: major histocompatibility complex; HLA: humanleukocyte antigen, or the human MHC gene locus; NGS: next-generationsequencing; PPV: positive predictive value; TSNA: tumor-specificneoantigen; FFPE: formalin-fixed, paraffin-embedded; NMD:nonsense-mediated decay; NSCLC: non-small-cell lung cancer; DC:dendritic cell.

It should be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Unless specifically stated or otherwise apparent from context, as usedherein the term “about” is understood as within a range of normaltolerance in the art, for example within 2 standard deviations of themean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unlessotherwise clear from context, all numerical values provided herein aremodified by the term about.

Any terms not directly defined herein shall be understood to have themeanings commonly associated with them as understood within the art ofthe invention. Certain terms are discussed herein to provide additionalguidance to the practitioner in describing the compositions, devices,methods and the like of aspects of the invention, and how to make or usethem. It will be appreciated that the same thing may be said in morethan one way. Consequently, alternative language and synonyms may beused for any one or more of the terms discussed herein. No significanceis to be placed upon whether or not a term is elaborated or discussedherein. Some synonyms or substitutable methods, materials and the likeare provided. Recital of one or a few synonyms or equivalents does notexclude use of other synonyms or equivalents, unless it is explicitlystated. Use of examples, including examples of terms, is forillustrative purposes only and does not limit the scope and meaning ofthe aspects of the invention herein.

All references, issued patents and patent applications cited within thebody of the specification are hereby incorporated by reference in theirentirety, for all purposes.

II. Methods of Identifying Antigens

Methods for identifying antigens (e.g., antigens derived from a tumor oran infectious disease organism) include identifying antigens that arelikely to be presented on a cell surface (e.g., presented by MHC on atumor cell, an infected cell, or an immune cell, including professionalantigen presenting cells such as dendritic cells), and/or are likely tobe immunogenic. As an example, one such method may comprise the stepsof: obtaining at least one of exome, transcriptome or whole genomenucleotide sequencing and/or expression data from a tumor, an infectedcell, or an infectious disease organism, wherein the nucleotidesequencing data and/or expression data is used to obtain datarepresenting peptide sequences of each of a set of antigens (e.g.,antigens derived from the tumor or infectious disease organism);inputting the peptide sequence of each antigen into one or morepresentation models to generate a set of numerical likelihoods that eachof the antigens is presented by one or more MHC alleles on a cellsurface, such as a tumor cell or an infected cell of the subject, theset of numerical likelihoods having been identified at least based onreceived mass spectrometry data; and selecting a subset of the set ofantigens based on the set of numerical likelihoods to generate a set ofselected antigens.

In one example directed to tumor vaccines, and which can be adapted toinfectious disease vaccines, the presentation model can comprise astatistical regression or a machine learning (e.g., deep learning) modeltrained on a set of reference data (also referred to as a training dataset) comprising a set of corresponding labels, wherein the set ofreference data is obtained from each of a plurality of distinct subjectswhere optionally some subjects can have a tumor, and wherein the set ofreference data comprises at least one of: data representing exomenucleic acid sequences from tumor tissue, data representing exomenucleic acid sequences from normal tissue, data representingtranscriptome nucleic acid sequences from tumor tissue, datarepresenting proteome sequences from tumor tissue, and data representingMHC peptidome sequences from tumor tissue, and data representing MHCpeptidome sequences from normal tissue. The reference data can furthercomprise mass spectrometry data, sequencing data, RNA sequencing data,expression profiling data, and proteomics data for single-allele celllines engineered to express a predetermined MHC allele that aresubsequently exposed to synthetic protein, normal and tumor human celllines, and fresh and frozen primary samples, and T cell assays (e.g.,ELISPOT). In certain aspects, the set of reference data includes eachform of reference data.

The presentation model can comprise a set of features derived at leastin part from the set of reference data, and wherein the set of featurescomprises at least one of allele dependent-features andallele-independent features. In certain aspects each feature isincluded.

Methods for identifying antigens also include generating an output forconstructing a personalized vaccine by identifying one or more antigensthat are likely to be presented on a surface of subject's cells, such asa tumor cell or infected cell. As an example directed to tumor vaccines,and which can be adapted to infectious disease vaccines, one such methodmay comprise the steps of: obtaining at least one of exome,transcriptome, or whole genome nucleotide sequencing and/or expressiondata from the tumor cells and normal cells of the subject, wherein thenucleotide sequencing and/or expression data is used to obtain datarepresenting peptide sequences of each of a set of antigens identifiedby comparing the nucleotide sequencing and/or expression data from thetumor cells and the nucleotide sequencing and/or expression data fromthe normal cells (e.g., in the case of neoantigens wherein the peptidesequence of each neoantigen comprises at least one alteration that makesit distinct from the corresponding wild-type peptide sequence or incases of antigens without a mutation where peptides are derived from anypolypeptide known to or have been found to have altered expression in atumor cell or cancerous tissue in comparison to a normal cell ortissue), peptide sequence identified from the normal cells of thesubject; encoding the peptide sequences of each of the antigens into acorresponding numerical vector, each numerical vector includinginformation regarding a plurality of amino acids that make up thepeptide sequence and a set of positions of the amino acids in thepeptide sequence; inputting the numerical vectors, using a computerprocessor, into a deep learning presentation model to generate a set ofpresentation likelihoods for the set of antigens, each presentationlikelihood in the set representing the likelihood that a correspondingantigen is presented by one or more class II MHC alleles on the surfaceof the tumor cells of the subject, the deep learning presentation model;selecting a subset of the set of antigens based on the set ofpresentation likelihoods to generate a set of selected antigens; andgenerating the output for constructing the personalized cancer vaccinebased on the set of selected antigens.

Specific methods for identifying antigens, including neoantigens, areknown to those skilled in the art, for example the methods described inmore detail in international patent application publicationsWO/2017/106638, WO/2018/195357, and WO/2018/208856, each hereinincorporated by reference, in their entirety, for all purposes.

A method of treating a subject having a tumor is disclosed herein,comprising performing the steps of any of the antigen identificationmethods described herein, and further comprising obtaining a tumorvaccine comprising the set of selected antigens, and administering thetumor vaccine to the subject.

A method disclosed herein can also include identifying one or more Tcells that are antigen-specific for at least one of the antigens in thesubset. In some embodiments, the identification comprises co-culturingthe one or more T cells with one or more of the antigens in the subsetunder conditions that expand the one or more antigen-specific T cells.In further embodiments, the identification comprises contacting the oneor more T cells with a tetramer comprising one or more of the antigensin the subset under conditions that allow binding between the T cell andthe tetramer. In even further embodiments, the method disclosed hereincan also include identifying one or more T cell receptors (TCR) of theone or more identified T cells. In certain embodiments, identifying theone or more T cell receptors comprises sequencing the T cell receptorsequences of the one or more identified T cells. The method disclosedherein can further comprise genetically engineering a plurality of Tcells to express at least one of the one or more identified T cellreceptors; culturing the plurality of T cells under conditions thatexpand the plurality of T cells; and infusing the expanded T cells intothe subject. In some embodiments, genetically engineering the pluralityof T cells to express at least one of the one or more identified T cellreceptors comprises cloning the T cell receptor sequences of the one ormore identified T cells into an expression vector; and transfecting eachof the plurality of T cells with the expression vector. In someembodiments, the method disclosed herein further comprises culturing theone or more identified T cells under conditions that expand the one ormore identified T cells; and infusing the expanded T cells into thesubject.

Also disclosed herein is an isolated T cell that is antigen-specific forat least one selected antigen in the subset.

Also disclosed herein is a methods for manufacturing a tumor vaccine,comprising the steps of: obtaining at least one of exome, transcriptomeor whole genome tumor nucleotide sequencing and/or expression data fromthe tumor cell of the subject, wherein the tumor nucleotide sequencingand/or expression data is used to obtain data representing peptidesequences of each of a set of antigens (e.g., in the case of neoantigenswherein the peptide sequence of each neoantigen comprises at least onealteration that makes it distinct from the corresponding wild-typepeptide sequence or in cases of antigens without a mutation wherepeptides are derived from any polypeptide known to or have been found tohave altered expression in a tumor cell or cancerous tissue incomparison to a normal cell or tissue); inputting the peptide sequenceof each antigen into one or more presentation models to generate a setof numerical likelihoods that each of the antigens is presented by oneor more MHC alleles on the tumor cell surface of the tumor cell of thesubject, the set of numerical likelihoods having been identified atleast based on received mass spectrometry data; and selecting a subsetof the set of antigens based on the set of numerical likelihoods togenerate a set of selected antigens; and producing or having produced atumor vaccine comprising the set of selected antigens.

Also disclosed herein is a tumor vaccine including a set of selectedantigens selected by performing the method comprising the steps of:obtaining at least one of exome, transcriptome or whole genome tumornucleotide sequencing and/or expression data from the tumor cell of thesubject, wherein the tumor nucleotide sequencing and/or expression datais used to obtain data representing peptide sequences of each of a setof antigens, and wherein the peptide sequence of each antigen (e.g., inthe case of neoantigens wherein the peptide sequence of each neoantigencomprises at least one alteration that makes it distinct from thecorresponding wild-type peptide sequence or in other cases of antigenswithout a mutation where peptides are derived from any polypeptide knownto or have been found to have altered expression in a tumor cell orcancerous tissue in comparison to a normal cell or tissue); inputtingthe peptide sequence of each antigen into one or more presentationmodels to generate a set of numerical likelihoods that each of theantigens is presented by one or more MHC alleles on the tumor cellsurface of the tumor cell of the subject, the set of numericallikelihoods having been identified at least based on received massspectrometry data; and selecting a subset of the set of antigens basedon the set of numerical likelihoods to generate a set of selectedantigens; and producing or having produced a tumor vaccine comprisingthe set of selected antigens.

The tumor vaccine may include one or more of a nucleic acid sequence, apolypeptide sequence, RNA, DNA, a cell, a plasmid, or a vector.

The tumor vaccine may include one or more antigens presented on thetumor cell surface.

The tumor vaccine may include one or more antigens that is immunogenicin the subject.

The tumor vaccine may not include one or more antigens that induce anautoimmune response against normal tissue in the subject.

The tumor vaccine may include an adjuvant.

The tumor vaccine may include an excipient.

A method disclosed herein may also include selecting antigens that havean increased likelihood of being presented on the tumor cell surfacerelative to unselected antigens based on the presentation model.

A method disclosed herein may also include selecting antigens that havean increased likelihood of being capable of inducing a tumor-specificimmune response in the subject relative to unselected antigens based onthe presentation model.

A method disclosed herein may also include selecting antigens that havean increased likelihood of being capable of being presented to naïve Tcells by professional antigen presenting cells (APCs) relative tounselected antigens based on the presentation model, optionally whereinthe APC is a dendritic cell (DC).

A method disclosed herein may also include selecting antigens that havea decreased likelihood of being subject to inhibition via central orperipheral tolerance relative to unselected antigens based on thepresentation model.

A method disclosed herein may also include selecting antigens that havea decreased likelihood of being capable of inducing an autoimmuneresponse to normal tissue in the subject relative to unselected antigensbased on the presentation model.

The exome or transcriptome nucleotide sequencing and/or expression datamay be obtained by performing sequencing on the tumor tissue.

The sequencing may be next generation sequencing (NGS) or any massivelyparallel sequencing approach.

The set of numerical likelihoods may be further identified by at leastMHC-allele interacting features comprising at least one of: thepredicted affinity with which the MHC allele and the antigen encodedpeptide bind; the predicted stability of the antigen encoded peptide-MHCcomplex; the sequence and length of the antigen encoded peptide; theprobability of presentation of antigen encoded peptides with similarsequence in cells from other individuals expressing the particular MHCallele as assessed by mass-spectrometry proteomics or other means; theexpression levels of the particular MHC allele in the subject inquestion (e.g. as measured by RNA-seq or mass spectrometry); the overallneoantigen encoded peptide-sequence-independent probability ofpresentation by the particular MHC allele in other distinct subjects whoexpress the particular MHC allele; the overall neoantigen encodedpeptide-sequence-independent probability of presentation by MHC allelesin the same family of molecules (e.g., HLA-A, HLA-B, HLA-C, HLA-DQ,HLA-DR, HLA-DP) in other distinct subjects.

The set of numerical likelihoods are further identified by at leastMHC-allele noninteracting features comprising at least one of: the C-and N-terminal sequences flanking the neoantigen encoded peptide withinits source protein sequence; the presence of protease cleavage motifs inthe neoantigen encoded peptide, optionally weighted according to theexpression of corresponding proteases in the tumor cells (as measured byRNA-seq or mass spectrometry); the turnover rate of the source proteinas measured in the appropriate cell type; the length of the sourceprotein, optionally considering the specific splice variants(“isoforms”) most highly expressed in the tumor cells as measured byRNA-seq or proteome mass spectrometry, or as predicted from theannotation of germline or somatic splicing mutations detected in DNA orRNA sequence data; the level of expression of the proteasome,immunoproteasome, thymoproteasome, or other proteases in the tumor cells(which may be measured by RNA-seq, proteome mass spectrometry, orimmunohistochemistry); the expression of the source gene of theneoantigen encoded peptide (e.g., as measured by RNA-seq or massspectrometry); the typical tissue-specific expression of the source geneof the neoantigen encoded peptide during various stages of the cellcycle; a comprehensive catalog of features of the source protein and/orits domains as can be found in e.g. uniProt or PDBwww.rcsb.org/pdb/home/home.do; features describing the properties of thedomain of the source protein containing the peptide, for example:secondary or tertiary structure (e.g., alpha helix vs beta sheet);alternative splicing; the probability of presentation of peptides fromthe source protein of the neoantigen encoded peptide in question inother distinct subjects; the probability that the peptide will not bedetected or over-represented by mass spectrometry due to technicalbiases; the expression of various gene modules/pathways as measured byRNASeq (which need not contain the source protein of the peptide) thatare informative about the state of the tumor cells, stroma, ortumor-infiltrating lymphocytes (TILs); the copy number of the sourcegene of the neoantigen encoded peptide in the tumor cells; theprobability that the peptide binds to the TAP or the measured orpredicted binding affinity of the peptide to the TAP; the expressionlevel of TAP in the tumor cells (which may be measured by RNA-seq,proteome mass spectrometry, immunohistochemistry); presence or absenceof tumor mutations, including, but not limited to: driver mutations inknown cancer driver genes such as EGFR, KRAS, ALK, RET, ROS1, TP53,CDKN2A, CDKN2B, NTRK1, NTRK2, NTRK3, and in genes encoding the proteinsinvolved in the antigen presentation machinery (e.g., B2M, HLA-A, HLA-B,HLA-C, TAP-1, TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, HLA-DMB,HLA-DO, HLA-DOA, HLA-DOB, HLA-DP, HLA-DPA1, HLA-DPB1, HLA-DQ, HLA-DQA1,HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DR, HLA-DRA, HLA-DRB1, HLA-DRB3,HLA-DRB4, HLA-DRB5 or any of the genes coding for components of theproteasome or immunoproteasome). Peptides whose presentation relies on acomponent of the antigen-presentation machinery that is subject toloss-of-function mutation in the tumor have reduced probability ofpresentation; presence or absence of functional germline polymorphisms,including, but not limited to: in genes encoding the proteins involvedin the antigen presentation machinery (e.g., B2M, HLA-A, HLA-B, HLA-C,TAP-1, TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, HLA-DMB, HLA-DO,HLA-DOA, HLA-DOB, HLA-DP, HLA-DPA1, HLA-DPB1, HLA-DQ, HLA-DQA1,HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DR, HLA-DRA, HLA-DRB1, HLA-DRB3,HLA-DRB4, HLA-DRB5 or any of the genes coding for components of theproteasome or immunoproteasome); tumor type (e.g., NSCLC, melanoma);clinical tumor subtype (e.g., squamous lung cancer vs. non-squamous);smoking history; the typical expression of the source gene of thepeptide in the relevant tumor type or clinical subtype, optionallystratified by driver mutation.

The at least one alteration may be a frameshift or nonframeshift indel,missense or nonsense substitution, splice site alteration, genomicrearrangement or gene fusion, or any genomic or expression alterationgiving rise to a neoORF.

The tumor cell may be selected from the group consisting of: lungcancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidneycancer, gastric cancer, colon cancer, testicular cancer, head and neckcancer, pancreatic cancer, brain cancer, B-cell lymphoma, acutemyelogenous leukemia, chronic myelogenous leukemia, chronic lymphocyticleukemia, and T cell lymphocytic leukemia, non-small cell lung cancer,and small cell lung cancer.

A method disclosed herein may also include obtaining a tumor vaccinecomprising the set of selected neoantigens or a subset thereof,optionally further comprising administering the tumor vaccine to thesubject.

At least one of neoantigens in the set of selected neoantigens, when inpolypeptide form, may include at least one of: a binding affinity withMHC with an IC50 value of less than 1000 nM, for MHC Class Ipolypeptides a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 aminoacids, for MHC Class II polypeptides a length of 6-30, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 amino acids, presence of sequence motifs within or near thepolypeptide in the parent protein sequence promoting proteasomecleavage, and presence of sequence motifs promoting TAP transport. ForMHC Class II, presence of sequence motifs within or near the peptidepromoting cleavage by extracellular or lysosomal proteases (e.g.,cathepsins) or HLA-DM catalyzed HLA binding.

Disclosed herein is are methods for identifying one or more neoantigensthat are likely to be presented on a tumor cell surface of a tumor cell,comprising executing the steps of: receiving mass spectrometry datacomprising data associated with a plurality of isolated peptides elutedfrom major histocompatibility complex (MHC) derived from a plurality offresh or frozen tumor samples; obtaining a training data set by at leastidentifying a set of training peptide sequences present in the tumorsamples and presented on one or more MHC alleles associated with eachtraining peptide sequence; obtaining a set of training protein sequencesbased on the training peptide sequences; and training a set of numericalparameters of a presentation model using the training protein sequencesand the training peptide sequences, the presentation model providing aplurality of numerical likelihoods that peptide sequences from the tumorcell are presented by one or more MHC alleles on the tumor cell surface.

The presentation model may represent dependence between: presence of apair of a particular one of the MHC alleles and a particular amino acidat a particular position of a peptide sequence; and likelihood ofpresentation on the tumor cell surface, by the particular one of the MHCalleles of the pair, of such a peptide sequence comprising theparticular amino acid at the particular position.

A method disclosed herein can also include selecting a subset ofneoantigens, wherein the subset of neoantigens is selected because eachhas an increased likelihood that it is presented on the cell surface ofthe tumor relative to one or more distinct tumor neoantigens.

A method disclosed herein can also include selecting a subset ofneoantigens, wherein the subset of neoantigens is selected because eachhas an increased likelihood that it is capable of inducing atumor-specific immune response in the subject relative to one or moredistinct tumor neoantigens.

A method disclosed herein can also include selecting a subset ofneoantigens, wherein the subset of neoantigens is selected because eachhas an increased likelihood that it is capable of being presented tonaïve T cells by professional antigen presenting cells (APCs) relativeto one or more distinct tumor neoantigens, optionally wherein the APC isa dendritic cell (DC).

A method disclosed herein can also include selecting a subset ofneoantigens, wherein the subset of neoantigens is selected because eachhas a decreased likelihood that it is subject to inhibition via centralor peripheral tolerance relative to one or more distinct tumorneoantigens.

A method disclosed herein can also include selecting a subset ofneoantigens, wherein the subset of neoantigens is selected because eachhas a decreased likelihood that it is capable of inducing an autoimmuneresponse to normal tissue in the subject relative to one or moredistinct tumor neoantigens.

A method disclosed herein can also include selecting a subset ofneoantigens, wherein the subset of neoantigens is selected because eachhas a decreased likelihood that it will be differentiallypost-translationally modified in tumor cells versus APCs, optionallywherein the APC is a dendritic cell (DC).

The practice of the methods herein will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: MackPublishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry3^(rd) Ed. (Plenum Press) Vols A and B(1992).

III. Identification of Tumor Specific Mutations in Neoantigens

Also disclosed herein are methods for the identification of certainmutations (e.g., the variants or alleles that are present in cancercells). In particular, these mutations can be present in the genome,transcriptome, proteome, or exome of cancer cells of a subject havingcancer but not in normal tissue from the subject. Specific methods foridentifying neoantigens, including shared neoantigens, that are specificto tumors are known to those skilled in the art, for example the methodsdescribed in more detail in U.S. Pat. No. 10,055,540, US ApplicationPub. No. US20200010849A1, and international patent applicationpublications WO/2018/195357 and WO/2018/208856, each herein incorporatedby reference, in their entirety, for all purposes.

Genetic mutations in tumors can be considered useful for theimmunological targeting of tumors if they lead to changes in the aminoacid sequence of a protein exclusively in the tumor. Useful mutationsinclude: (1) non-synonymous mutations leading to different amino acidsin the protein; (2) read-through mutations in which a stop codon ismodified or deleted, leading to translation of a longer protein with anovel tumor-specific sequence at the C-terminus; (3) splice sitemutations that lead to the inclusion of an intron in the mature mRNA andthus a unique tumor-specific protein sequence; (4) chromosomalrearrangements that give rise to a chimeric protein with tumor-specificsequences at the junction of 2 proteins (i.e., gene fusion); (5)frameshift mutations or deletions that lead to a new open reading framewith a novel tumor-specific protein sequence. Mutations can also includeone or more of nonframeshift indel, missense or nonsense substitution,splice site alteration, genomic rearrangement or gene fusion, or anygenomic or expression alteration giving rise to a neoORF.

Peptides with mutations or mutated polypeptides arising from forexample, splice-site, frameshift, readthrough, or gene fusion mutationsin tumor cells can be identified by sequencing DNA, RNA or protein intumor versus normal cells.

Also mutations can include previously identified tumor specificmutations. Known tumor mutations can be found at the Catalogue ofSomatic Mutations in Cancer (COSMIC) database.

A variety of methods are available for detecting the presence of aparticular mutation or allele in an individual's DNA or RNA.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. For example, several techniques have beendescribed including dynamic allele-specific hybridization (DASH),microplate array diagonal gel electrophoresis (MADGE), pyrosequencing,oligonucleotide-specific ligation, the TaqMan system as well as variousDNA “chip” technologies such as the Affymetrix SNP chips. These methodsutilize amplification of a target genetic region, typically by PCR.Still other methods, based on the generation of small signal moleculesby invasive cleavage followed by mass spectrometry or immobilizedpadlock probes and rolling-circle amplification. Several of the methodsknown in the art for detecting specific mutations are summarized below.

PCR based detection means can include multiplex amplification of aplurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

Several methods have been developed to facilitate analysis of singlenucleotide polymorphisms in genomic DNA or cellular RNA. For example, asingle base polymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R.(U.S. Pat. No. 4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide(s) present in the polymorphic site of the target molecule iscomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

A solution-based method can be used for determining the identity of anucleotide of a polymorphic site. Cohen, D. et al. (French Patent2,650,840; PCT Appln. No. WO91/02087). As in the Mundy method of U.S.Pat. No. 4,656,127, a primer is employed that is complementary toallelic sequences immediately 3′ to a polymorphic site. The methoddetermines the identity of the nucleotide of that site using labeleddideoxynucleotide derivatives, which, if complementary to the nucleotideof the polymorphic site will become incorporated onto the terminus ofthe primer.

An alternative method, known as Genetic Bit Analysis or GBA is describedby Goelet, P. et al. (PCT Appln. No. 92/15712). The method of Goelet, P.et al. uses mixtures of labeled terminators and a primer that iscomplementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. can be a heterogeneous phase assay, in which theprimer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assayingpolymorphic sites in DNA have been described (Komher, J. S. et al.,Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res.18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA in that they utilizeincorporation of labeled deoxynucleotides to discriminate between basesat a polymorphic site. In such a format, since the signal isproportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

A number of initiatives obtain sequence information directly frommillions of individual molecules of DNA or RNA in parallel. Real-timesingle molecule sequencing-by-synthesis technologies rely on thedetection of fluorescent nucleotides as they are incorporated into anascent strand of DNA that is complementary to the template beingsequenced. In one method, oligonucleotides 30-50 bases in length arecovalently anchored at the 5′ end to glass cover slips. These anchoredstrands perform two functions. First, they act as capture sites for thetarget template strands if the templates are configured with capturetails complementary to the surface-bound oligonucleotides. They also actas primers for the template directed primer extension that forms thebasis of the sequence reading. The capture primers function as a fixedposition site for sequence determination using multiple cycles ofsynthesis, detection, and chemical cleavage of the dye-linker to removethe dye. Each cycle consists of adding the polymerase/labeled nucleotidemixture, rinsing, imaging and cleavage of dye. In an alternative method,polymerase is modified with a fluorescent donor molecule and immobilizedon a glass slide, while each nucleotide is color-coded with an acceptorfluorescent moiety attached to a gamma-phosphate. The system detects theinteraction between a fluorescently-tagged polymerase and afluorescently modified nucleotide as the nucleotide becomes incorporatedinto the de novo chain. Other sequencing-by-synthesis technologies alsoexist.

Any suitable sequencing-by-synthesis platform can be used to identifymutations. As described above, four major sequencing-by-synthesisplatforms are currently available: the Genome Sequencers from Roche/454Life Sciences, the 1G Analyzer from Illumina/Solexa, the SOLiD systemfrom Applied BioSystems, and the Heliscope system from HelicosBiosciences. Sequencing-by-synthesis platforms have also been describedby Pacific BioSciences and VisiGen Biotechnologies. In some embodiments,a plurality of nucleic acid molecules being sequenced is bound to asupport (e.g., solid support). To immobilize the nucleic acid on asupport, a capture sequence/universal priming site can be added at the3′ and/or 5′ end of the template. The nucleic acids can be bound to thesupport by hybridizing the capture sequence to a complementary sequencecovalently attached to the support. The capture sequence (also referredto as a universal capture sequence) is a nucleic acid sequencecomplementary to a sequence attached to a support that may dually serveas a universal primer.

As an alternative to a capture sequence, a member of a coupling pair(such as, e.g., antibody/antigen, receptor/ligand, or the avidin-biotinpair as described in, e.g., US Patent Application No. 2006/0252077) canbe linked to each fragment to be captured on a surface coated with arespective second member of that coupling pair.

Subsequent to the capture, the sequence can be analyzed, for example, bysingle molecule detection/sequencing, e.g., as described in the Examplesand in U.S. Pat. No. 7,283,337, including template-dependentsequencing-by-synthesis. In sequencing-by-synthesis, the surface-boundmolecule is exposed to a plurality of labeled nucleotide triphosphatesin the presence of polymerase. The sequence of the template isdetermined by the order of labeled nucleotides incorporated into the 3′end of the growing chain. This can be done in real time or can be donein a step-and-repeat mode. For real-time analysis, different opticallabels to each nucleotide can be incorporated and multiple lasers can beutilized for stimulation of incorporated nucleotides.

Sequencing can also include other massively parallel sequencing or nextgeneration sequencing (NGS) techniques and platforms. Additionalexamples of massively parallel sequencing techniques and platforms arethe Illumina HiSeq or MiSeq, Thermo PGM or Proton, the Pac Bio RS II orSequel, Qiagen's Gene Reader, and the Oxford Nanopore MinION. Additionalsimilar current massively parallel sequencing technologies can be used,as well as future generations of these technologies.

Any cell type or tissue can be utilized to obtain nucleic acid samplesfor use in methods described herein. For example, a DNA or RNA samplecan be obtained from a tumor or a bodily fluid, e.g., blood, obtained byknown techniques (e.g. venipuncture) or saliva. Alternatively, nucleicacid tests can be performed on dry samples (e.g. hair or skin). Inaddition, a sample can be obtained for sequencing from a tumor andanother sample can be obtained from normal tissue for sequencing wherethe normal tissue is of the same tissue type as the tumor. A sample canbe obtained for sequencing from a tumor and another sample can beobtained from normal tissue for sequencing where the normal tissue is ofa distinct tissue type relative to the tumor.

Tumors can include one or more of lung cancer, melanoma, breast cancer,ovarian cancer, prostate cancer, kidney cancer, gastric cancer, coloncancer, testicular cancer, head and neck cancer, pancreatic cancer,brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, and T celllymphocytic leukemia, non-small cell lung cancer, and small cell lungcancer.

Alternatively, protein mass spectrometry can be used to identify orvalidate the presence of mutated peptides bound to MHC proteins on tumorcells. Peptides can be acid-eluted from tumor cells or from HLAmolecules that are immunoprecipitated from tumor, and then identifiedusing mass spectrometry.

IV. Antigens

Antigens can include nucleotides or polypeptides. For example, anantigen can be an RNA sequence that encodes for a polypeptide sequence.Antigens useful in vaccines can therefore include nucleic acid sequencesor polypeptide sequences.

Disclosed herein are isolated peptides that comprise tumor specificmutations identified by the methods disclosed herein, peptides thatcomprise known tumor specific mutations, and mutant polypeptides orfragments thereof identified by methods disclosed herein. Neoantigenpeptides can be described in the context of their coding sequence wherea neoantigen includes the nucleic acid sequence (e.g., DNA or RNA) thatcodes for the related polypeptide sequence.

Also disclosed herein are peptides derived from any polypeptide known toor have been found to have altered expression in a tumor cell orcancerous tissue in comparison to a normal cell or tissue, for exampleany polypeptide known to or have been found to be aberrantly expressedin a tumor cell or cancerous tissue in comparison to a normal cell ortissue. Suitable polypeptides from which the antigenic peptides can bederived can be found for example in the COSMIC database. COSMIC curatescomprehensive information on somatic mutations in human cancer. Thepeptide contains the tumor specific mutation.

The modified adenoviral vectors and other constructs described hereincan be used to deliver antigens from any organism, including theirtoxins or other by-products, to prevent and/or treat infection or otheradverse reactions associated with the organism or its by-product.

Antigens that can be incorporated into a vaccine (e.g., encoded in acassette) include immunogens which are useful to immunize a human ornon-human animal against viruses, such as pathogenic viruses whichinfect human and non-human vertebrates. Antigens may be selected from avariety of viral families. Example of desirable viral families againstwhich an immune response would be desirable include, the picornavirusfamily, which includes the genera rhinoviruses, which are responsiblefor about 50% of cases of the common cold; the genera enteroviruses,which include polioviruses, coxsackieviruses, echoviruses, and humanenteroviruses such as hepatitis A virus; and the genera apthoviruses,which are responsible for foot and mouth diseases, primarily innon-human animals. Within the picornavirus family of viruses, targetantigens include the VP1, VP2, VP3, VP4, and VPG. Another viral familyincludes the calcivirus family, which encompasses the Norwalk group ofviruses, which are an important causative agent of epidemicgastroenteritis. Still another viral family desirable for use intargeting antigens for inducing immune responses in humans and non-humananimals is the togavirus family, which includes the genera alphavirus,which include Sindbis viruses, RossRiver virus, and Venezuelan, Eastern& Western Equine encephalitis, and rubivirus, including Rubella virus.The Flaviviridae family includes dengue, yellow fever, Japaneseencephalitis, St. Louis encephalitis and tick borne encephalitisviruses. Other target antigens may be generated from the Hepatitis C orthe coronavirus family, which includes a number of non-human virusessuch as infectious bronchitis virus (poultry), porcine transmissiblegastroenteric virus (pig), porcine hemagglutinating encephalomyelitisvirus (pig), feline infectious peritonitis virus (cats), feline entericcoronavirus (cat), canine coronavirus (dog), and human respiratorycoronaviruses, which may cause the common cold and/or non-A, B or Chepatitis. Within the coronavirus family, target antigens include the E1(also called M or matrix protein), E2 (also called S or Spike protein),E3 (also called HE or hemagglutin-elterose) glycoprotein (not present inall coronaviruses), or N (nucleocapsid). Still other antigens may betargeted against the rhabdovirus family, which includes the generavesiculovirus (e.g., Vesicular Stomatitis Virus), and the generallyssavirus (e.g., rabies). Within the rhabdovirus family, suitableantigens may be derived from the G protein or the N protein. The familyfiloviridae, which includes hemorrhagic fever viruses such as Marburgand Ebola virus, may be a suitable source of antigens. The paramyxovirusfamily includes parainfluenza Virus Type 1, parainfluenza Virus Type 3,bovine parainfluenza Virus Type 3, rubulavirus (mumps virus),parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastledisease virus (chickens), rinderpest, morbillivirus, which includesmeasles and canine distemper, and pneumovirus, which includesrespiratory syncytial virus (e.g., the glyco-(G) protein and the fusion(F) protein, for which sequences are available from GenBank). Influenzavirus is classified within the family orthomyxovirus and can be suitablesource of antigens (e.g., the HA protein, the N1 protein). Thebunyavirus family includes the genera bunyavirus (Californiaencephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus(puremala is a hemahagin fever virus), nairovirus (Nairobi sheepdisease) and various unassigned bungaviruses. The arenavirus familyprovides a source of antigens against LCM and Lassa fever virus. Thereovirus family includes the genera reovirus, rotavirus (which causesacute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue). The retrovirus family includes the sub-family oncorivirinalwhich encompasses such human and veterinary diseases as feline leukemiavirus, HTLVI and HTLVII, lentivirinal (which includes humanimmunodeficiency virus (HIV), simian immunodeficiency virus (SIV),feline immunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp41, or smaller fragments thereof,e.g., of at least about 8 amino acids in length. Similarly, fragments ofthe tat protein may be selected. [See, U.S. Pat. Nos. 5,891,994 and6,193,981.] See, also, the HIV and SIV proteins described in D. H.Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R. R. Amara,et al, Science, 292:69-74 (6 Apr. 2001). In another example, the HIVand/or SIV immunogenic proteins or peptides may be used to form fusionproteins or other immunogenic molecules. See, e.g., the HIV-1 Tat and/orNef fusion proteins and immunization regimens described in WO 01/54719,published Aug. 2, 2001, and WO 99/16884, published Apr. 8, 1999. Theinvention is not limited to the HIV and/or SIV immunogenic proteins orpeptides described herein. In addition, a variety of modifications tothese proteins have been described or could readily be made by one ofskill in the art. See, e.g., the modified gag protein that is describedin U.S. Pat. No. 5,972,596. Further, any desired HIV and/or SIVimmunogens may be delivered alone or in combination. Such combinationsmay include expression from a single vector or from multiple vectors.The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/orenteritis. The parvovirus family feline parvovirus (feline enteritis),feline panleucopeniavirus, canine parvovirus, and porcine parvovirus.The herpesvirus family includes the sub-family alphaherpesvirinae, whichencompasses the genera simplexvirus (HSVI, HSVII), varicellovirus(pseudorabies, varicella zoster) and the sub-family betaherpesvirinae,which includes the genera cytomegalovirus (Human CMV), muromegalovirus)and the sub-family gammaherpesvirinae, which includes the generalymphocryptovirus, EBV (Burkitts lymphoma), infectious rhinotracheitis,Marek's disease virus, and rhadinovirus. The poxvirus family includesthe sub-family chordopoxyirinae, which encompasses the generaorthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus,avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and thesub-family entomopoxyirinae. The hepadnavirus family includes theHepatitis B virus. One unclassified virus which may be suitable sourceof antigens is the Hepatitis delta virus. Still other viral sources mayinclude avian infectious bursal disease virus and porcine respiratoryand reproductive syndrome virus. The alphavirus family includes equinearteritis virus and various Encephalitis viruses.

Antigens that can be incorporated into a vaccine (e.g., encoded in acassette) also include immunogens which are useful to immunize a humanor non-human animal against pathogens including bacteria, fungi,parasitic microorganisms or multicellular parasites which infect humanand non-human vertebrates. Examples of bacterial pathogens includepathogenic gram-positive cocci include pneumococci; staphylococci; andstreptococci. Pathogenic gram-negative cocci include meningococcus;gonococcus. Pathogenic enteric gram-negative bacilli includeenterobacteriaceae; pseudomonas, acinetobacteria and eikenella;melioidosis; salmonella; shigella; haemophilus (Haemophilus influenzae,Haemophilus somnus); moraxella; H. ducreyi (which causes chancroid);brucella; Franisella tularensis (which causes tularemia); yersinia(pasteurella); streptobacillus moniliformis and spirillum. Gram-positivebacilli include Listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Examples of specific bacterium species are, withoutlimitation, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Moraxella catarrhalis,Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae,Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci,Bordetella pertussis, Salmonella typhi, Salmonella typhimurium,Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae,Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacteriumavium, Mycobacterium intracellulare complex, Proteus mirabilis, Proteusvulgaris, Staphylococcus aureus, Clostridium tetani, Leptospirainterrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurellamultocida, Actinobacillus pleuropneumoniae and Mycoplasma gallisepticum.Pathogenic spirochetal diseases include syphilis; treponematoses: yaws,pinta and endemic syphilis; and leptospirosis. Other infections causedby higher pathogen bacteria and pathogenic fungi include actinomycosis;nocardiosis; cryptococcosis (Cryptococcus), blastomycosis (Blastomyces),histoplasmosis (Histoplasma) and coccidioidomycosis (Coccidiodes);candidiasis (Candida), aspergillosis (Aspergillis), and mucormycosis;sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis,mycetoma and chromomycosis; and dermatophytosis. Rickettsial infectionsinclude Typhus fever, Rocky Mountain spotted fever, Q fever, andRickettsialpox. Examples of mycoplasma and chlamydial infectionsinclude: Mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis;and perinatal chlamydial infections. Pathogenic eukaryotes encompasspathogenic protozoans and helminths and infections produced therebyinclude: amebiasis; malaria; leishmaniasis (e.g., caused by Leishmaniamajor); trypanosomiasis; toxoplasmosis (e.g., caused by Toxoplasmagondii); Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis;giardiasis (e.g., caused by Giardia); trichinosis (e.g., caused byTrichomonas); filariasis; schistosomiasis (e.g., caused by Schistosoma);nematodes; trematodes or flukes; and cestode (tapeworm) infections.Other parasitic infections may be caused by Ascaris, Trichuris,Cryptosporidium, and Pneumocystis carinii, among others.

Also disclosed herein are peptides derived from any polypeptideassociated with an infectious disease organism, an infection in asubject, or an infected cell of a subject. Antigens can be derived fromnucleic acid sequences or polypeptide sequences of an infectious diseaseorganism. Polypeptide sequences of an infectious disease organisminclude, but are not limited to, a pathogen-derived peptide, avirus-derived peptide, a bacteria-derived peptide, a fungus-derivedpeptide, and/or a parasite-derived peptide. Infectious disease organisminclude, but are not limited to, Severe acute respiratorysyndrome-related coronavirus (SARS), severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2), Ebola, HIV, Hepatitis B virus (HBV),influenza, Hepatitis C virus (HCV), and tuberculosis.

Antigens can be selected that are predicted to be presented on the cellsurface of a cell, such as a tumor cell, an infected cell, or an immunecell, including professional antigen presenting cells such as dendriticcells. Antigens can be selected that are predicted to be immunogenic.

One or more polypeptides encoded by an antigen nucleic acid sequence cancomprise at least one of: a binding affinity with MHC with an IC50 valueof less than 1000 nM, for MHC Class I peptides a length of 8-15, 8, 9,10, 11, 12, 13, 14, or 15 amino acids, presence of sequence motifswithin or near the peptide promoting proteasome cleavage, and presenceor sequence motifs promoting TAP transport. For MHC Class II peptides alength 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, presence of sequencemotifs within or near the peptide promoting cleavage by extracellular orlysosomal proteases (e.g., cathepsins) or HLA-DM catalyzed HLA binding.

One or more antigens can be presented on the surface of a tumor. One ormore antigens can be presented on the surface of an infected cell.

One or more antigens can be immunogenic in a subject having a tumor,e.g., capable of eliciting a T cell response or a B cell response in thesubject. One or more antigens can be immunogenic in a subject having orsuspected to have an infection, e.g., capable of eliciting a T cellresponse or a B cell response in the subject. One or more antigens canbe immunogenic in a subject at risk of an infection, e.g., capable ofeliciting a T cell response or a B cell response in the subject thatprovides immunological protection (i.e., immunity) against theinfection, e.g., such as stimulating the production of memory T cells,memory B cells, or antibodies specific to the infection.

One or more antigens can be capable of eliciting a B cell response, suchas the production of antibodies that recognize the one or more antigens.Antibodies can recognize linear polypeptide sequences or recognizesecondary and tertiary structures. Accordingly, B cell antigens caninclude linear polypeptide sequences or polypeptides having secondaryand tertiary structures, including, but not limited to, full-lengthproteins, protein subunits, protein domains, or any polypeptide sequenceknown or predicted to have secondary and tertiary structures.

One or more antigens that induce an autoimmune response in a subject canbe excluded from consideration in the context of vaccine generation fora subject.

The size of at least one antigenic peptide molecule (e.g., an epitopesequence) can comprise, but is not limited to, about 5, about 6, about7, about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 60, about 70, about 80, about 90, about 100, about 110,about 120 or greater amino molecule residues, and any range derivabletherein. In specific embodiments the antigenic peptide molecules areequal to or less than 50 amino acids.

Antigenic peptides and polypeptides can be: for MHC Class 115 residuesor less in length and usually consist of between about 8 and about 11residues, particularly 9 or 10 residues; for MHC Class II, 6-30residues, inclusive.

If desirable, a longer peptide can be designed in several ways. In onecase, when presentation likelihoods of peptides on HLA alleles arepredicted or known, a longer peptide could consist of either: (1)individual presented peptides with an extensions of 2-5 amino acidstoward the N- and C-terminus of each corresponding gene product; (2) aconcatenation of some or all of the presented peptides with extendedsequences for each. In another case, when sequencing reveals a long (>10residues) neoepitope sequence present in the tumor (e.g. due to aframeshift, read-through or intron inclusion that leads to a novelpeptide sequence), a longer peptide would consist of: (3) the entirestretch of novel tumor-specific or infectious disease-specific aminoacids—thus bypassing the need for computational or in vitro test-basedselection of the strongest HLA-presented shorter peptide. In both cases,use of a longer peptide allows endogenous processing by patient cellsand may lead to more effective antigen presentation and induction of Tcell responses.

Antigenic peptides and polypeptides can be presented on an HLA protein.In some aspects antigenic peptides and polypeptides are presented on anHLA protein with greater affinity than a wild-type peptide. In someaspects, an antigenic peptide or polypeptide can have an IC50 of atleast less than 5000 nM, at least less than 1000 nM, at least less than500 nM, at least less than 250 nM, at least less than 200 nM, at leastless than 150 nM, at least less than 100 nM, at least less than 50 nM orless.

In some aspects, antigenic peptides and polypeptides do not induce anautoimmune response and/or invoke immunological tolerance whenadministered to a subject.

Also provided are compositions comprising at least two or more antigenicpeptides. In some embodiments the composition contains at least twodistinct peptides. At least two distinct peptides can be derived fromthe same polypeptide. By distinct polypeptides is meant that the peptidevary by length, amino acid sequence, or both. The peptides can bederived from any polypeptide known to or have been found to contain atumor specific mutation or peptides derived from any polypeptide knownto or have been found to have altered expression in a tumor cell orcancerous tissue in comparison to a normal cell or tissue, for exampleany polypeptide known to or have been found to be aberrantly expressedin a tumor cell or cancerous tissue in comparison to a normal cell ortissue. Suitable polypeptides from which the antigenic peptides can bederived can be found for example in the COSMIC database or the AACRGenomics Evidence Neoplasia Information Exchange (GENIE) database.COSMIC curates comprehensive information on somatic mutations in humancancer. AACR GENIE aggregates and links clinical-grade cancer genomicdata with clinical outcomes from tens of thousands of cancer patients.In some aspects the tumor specific mutation is a driver mutation for aparticular cancer type. The peptides can be derived from any polypeptideknown to or suspected to be associated with an infectious diseaseorganism, or peptides derived from any polypeptide known to or have beenfound to have altered expression in an infected cell in comparison to anormal cell or tissue (e.g., an infectious disease polynucleotide orpolypeptide, including infectious disease polynucleotides orpolypeptides with expression restricted to a host cell).

Antigenic peptides and polypeptides having a desired activity orproperty can be modified to provide certain desired attributes, e.g.,improved pharmacological characteristics, while increasing or at leastretaining substantially all of the biological activity of the unmodifiedpeptide to bind the desired MHC molecule and activate the appropriate Tcell. For instance, antigenic peptide and polypeptides can be subject tovarious changes, such as substitutions, either conservative ornon-conservative, where such changes might provide for certainadvantages in their use, such as improved MHC binding, stability orpresentation. By conservative substitutions is meant replacing an aminoacid residue with another which is biologically and/or chemicallysimilar, e.g., one hydrophobic residue for another, or one polar residuefor another. The substitutions include combinations such as Gly, Ala;Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe,Tyr. The effect of single amino acid substitutions may also be probedusing D-amino acids. Such modifications can be made using well knownpeptide synthesis procedures, as described in e.g., Merrifield, Science232:341-347 (1986), Barany & Merrifield, The Peptides, Gross &Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart &Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed.(1984).

Modifications of peptides and polypeptides with various amino acidmimetics or unnatural amino acids can be particularly useful inincreasing the stability of the peptide and polypeptide in vivo.Stability can be assayed in a number of ways. For instance, peptidasesand various biological media, such as human plasma and serum, have beenused to test stability. See, e.g., Verhoef et al., Eur. J. Drug MetabPharmacokin. 11:291-302 (1986). Half-life of the peptides can beconveniently determined using a 25% human serum (v/v) assay. Theprotocol is generally as follows. Pooled human serum (Type AB, non-heatinactivated) is delipidated by centrifugation before use. The serum isthen diluted to 25% with RPMI tissue culture media and used to testpeptide stability. At predetermined time intervals a small amount ofreaction solution is removed and added to either 6% aqueoustrichloracetic acid or ethanol. The cloudy reaction sample is cooled (4degrees C.) for 15 minutes and then spun to pellet the precipitatedserum proteins. The presence of the peptides is then determined byreversed-phase HPLC using stability-specific chromatography conditions.

The peptides and polypeptides can be modified to provide desiredattributes other than improved serum half-life. For instance, theability of the peptides to induce CTL activity can be enhanced bylinkage to a sequence which contains at least one epitope that iscapable of inducing a T helper cell response. Immunogenic peptides/Thelper conjugates can be linked by a spacer molecule. The spacer istypically comprised of relatively small, neutral molecules, such asamino acids or amino acid mimetics, which are substantially unchargedunder physiological conditions. The spacers are typically selected from,e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids orneutral polar amino acids. It will be understood that the optionallypresent spacer need not be comprised of the same residues and thus canbe a hetero- or homo-oligomer. When present, the spacer will usually beat least one or two residues, more usually three to six residues.Alternatively, the peptide can be linked to the T helper peptide withouta spacer.

An antigenic peptide can be linked to the T helper peptide eitherdirectly or via a spacer either at the amino or carboxy terminus of thepeptide. The amino terminus of either the antigenic peptide or the Thelper peptide can be acylated. Exemplary T helper peptides includetetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite382-398 and 378-389.

Proteins or peptides can be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and can be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases located at theNational Institutes of Health website. The coding regions for knowngenes can be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

In a further aspect an antigen includes a nucleic acid (e.g.polynucleotide) that encodes an antigenic peptide or portion thereof.The polynucleotide can be, e.g., DNA, cDNA, PNA, CNA, RNA (e.g., mRNA),either single- and/or double-stranded, or native or stabilized forms ofpolynucleotides, such as, e.g., polynucleotides with a phosphorothioatebackbone, or combinations thereof and it may or may not contain introns.A still further aspect provides an expression vector capable ofexpressing a polypeptide or portion thereof. Expression vectors fordifferent cell types are well known in the art and can be selectedwithout undue experimentation. Generally, DNA is inserted into anexpression vector, such as a plasmid, in proper orientation and correctreading frame for expression. If necessary, DNA can be linked to theappropriate transcriptional and translational regulatory control nucleicacid sequences recognized by the desired host, although such controlsare generally available in the expression vector. The vector is thenintroduced into the host through standard techniques. Guidance can befound e.g. in Sambrook et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

V. Delivery Compositions

Also disclosed herein is an immunogenic composition, e.g., a vaccinecomposition, capable of raising a specific immune response, e.g., atumor-specific immune response or an infectious diseaseorganism-specific immune response. Vaccine compositions typicallycomprise one or a plurality of antigens, e.g., selected using a methoddescribed herein. Vaccine compositions can also be referred to asvaccines.

A vaccine can contain between 1 and 30 peptides, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 different peptides, 6, 7, 8, 9, 10 11, 12, 13, or 14different peptides, or 12, 13 or 14 different peptides. Peptides caninclude post-translational modifications. A vaccine can contain between1 and 100 or more nucleic acid sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or more different nucleic acid sequences, 6, 7, 8, 9, 10 11, 12, 13, or14 different nucleic acid sequences, or 12, 13 or 14 different nucleicacid sequences. A vaccine can contain between 1 and 30 antigensequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different antigensequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen sequences,or 12, 13 or 14 different antigen sequences.

A vaccine can contain between 1 and 30 antigen-encoding nucleic acidsequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more differentantigen-encoding nucleic acid sequences, 6, 7, 8, 9, 10 11, 12, 13, or14 different antigen-encoding nucleic acid sequences, or 12, 13 or 14different antigen-encoding nucleic acid sequences. Antigen-encodingnucleic acid sequences can refer to the antigen encoding portion of anantigen “cassette.” Features of an cassette are described in greaterdetail below.

A vaccine can contain between 1 and 30 distinct epitope-encoding nucleicacid sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more distinctepitope-encoding nucleic acid sequences, 6, 7, 8, 9, 10 11, 12, 13, or14 distinct epitope-encoding nucleic acid sequences, or 12, 13 or 14distinct epitope-encoding nucleic acid sequences. Epitope-encodingnucleic acid sequences can refer to sequences for individual epitopesequences.

A vaccine can contain at least two repeats of an epitope-encodingnucleic acid sequence. A used herein, a “repeat” refers to two or moreiterations of an identical nucleic acid epitope-encoding nucleic acidsequence (inclusive of the optional 5′ linker sequence and/or theoptional 3′ linker sequences described herein) within anantigen-encoding nucleic acid sequence. In one example, theantigen-encoding nucleic acid sequence portion of a cassette encodes atleast two repeats of an epitope-encoding nucleic acid sequence. Infurther non-limiting examples, the antigen-encoding nucleic acidsequence portion of a cassette encodes more than one distinct epitope,and at least one of the distinct epitopes is encoded by at least tworepeats of the nucleic acid sequence encoding the distinct epitope(i.e., at least two distinct epitope-encoding nucleic acid sequences).In illustrative non-limiting examples, an antigen-encoding nucleic acidsequence encodes epitopes A, B, and C encoded by epitope-encodingnucleic acid sequences epitope-encoding sequence A (E_(A)),epitope-encoding sequence B (E_(B)), and epitope-encoding sequence C(E_(C)), and exemplary antigen-encoding nucleic acid sequences havingrepeats of at least one of the distinct epitopes are illustrated by, butis not limited to, the formulas below:

-   -   Repeat of one distinct epitope (repeat of epitope A):    -   E_(A)-E_(B)-E_(C)-E_(A); or    -   E_(A)-E_(A)-E_(B)-E_(C)    -   Repeat of multiple distinct epitopes (repeats of epitopes A, B,        and C):    -   E_(A)-E_(B)-E_(C)-E_(A)-E_(B)-E_(C); or    -   E_(A)-E_(A)-E_(B)-E_(B)-E_(C)-E_(C)    -   Multiple repeats of multiple distinct epitopes (repeats of        epitopes A, B, and C):    -   E_(A)-E_(B)-E_(C)-E_(A)-E_(B)-E_(C)-E_(A)-E_(B)-E_(C); or    -   E_(A)-E_(A)-E_(A)-E_(B)-E_(B)-E_(B)-E_(C)-E_(C)-E_(C)

The above examples are not limiting and the antigen-encoding nucleicacid sequences having repeats of at least one of the distinct epitopescan encode each of the distinct epitopes in any order or frequency. Forexample, the order and frequency can be a random arrangement of thedistinct epitopes, e.g., in an example with epitopes A, B, and C, by theformulaE_(A)-E_(B)-E_(C)-E_(C)-E_(A)-E_(B)-E_(A)-E_(C)-E_(A)-E_(C)-E_(C)-E_(B).

Also provided for herein is an antigen-encoding cassette, theantigen-encoding cassette having at least one antigen-encoding nucleicacid sequence described, from 5′ to 3′ by the formula:

(E_(x)−(E^(N) _(n))_(y))_(z)

where E represents a nucleic acid sequence comprising at least one ofthe at least one distinct epitope-encoding nucleic acid sequences,

-   -   n represents the number of separate distinct epitope-encoding        nucleic acid sequences and is any integer including 0,    -   E^(N) represents a nucleic acid sequence comprising the separate        distinct epitope-encoding nucleic acid sequence for each        corresponding n,    -   for each iteration of z: x=0 or 1, y=0 or 1 for each n, and at        least one of x or y=1, and z=2 or greater, wherein the        antigen-encoding nucleic acid sequence comprises at least two        iterations of E, a given E^(N), or a combination thereof.

Each E or E^(N) can independently comprise any epitope-encoding nucleicacid sequence described herein. For example, Each E or E^(N) canindependently comprises a nucleic acid sequence described, from 5′ to3′, by the formula (L5_(b)-N_(c)-L3_(d)), where N comprises the distinctepitope-encoding nucleic acid sequence associated with each E or E^(N),where c=1, L5 comprises a 5′ linker sequence, where b=0 or 1, and L3comprises a 3′ linker sequence, where d=0 or 1. Epitopes and linkersthat can be used are further described herein, e.g., see “V. A.Cassettes” section.

Repeats of an epitope-encoding nucleic acid sequences (inclusive ofoptional 5′ linker sequence and/or the optional 3′ linker sequences) canbe linearly linked directly to one another (e.g., E_(A)-E_(A)- . . . asillustrated above). Repeats of an epitope-encoding nucleic acidsequences can be separated by one or more additional nucleotidessequences. In general, repeats of an epitope-encoding nucleic acidsequences can be separated by any size nucleic acid sequence applicablefor the compositions described herein. In one example, repeats of anepitope-encoding nucleic acid sequences can be separated by a separatedistinct epitope-encoding nucleic acid sequence (e.g.,E_(A)-E_(B)-E_(C)-E_(A) . . . , as illustrated above). In examples whererepeats are separated by a single separate distinct epitope-encodingnucleic acid sequence, and each epitope-encoding nucleic acid sequences(inclusive of optional 5′ linker sequence and/or the optional 3′ linkersequences) encodes a peptide 25 amino acids in length, the repeats canbe separated by 75 nucleotides, such as in antigen-encoding nucleic acidrepresented by E_(A)-E_(B)-E_(A) . . . , E_(A) is separated by 75nucleotides. In an illustrative example, an antigen-encoding nucleicacid having the sequence

(SEQ ID NO: 74) VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTencoding repeats of 25mer antigens Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQ (SEQID NO: 75)) and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 76)), therepeats of Trp1 are separated by the 25mer Trp2 and thus the repeats ofthe Trp1 epitope-encoding nucleic acid sequences are separated the 75nucleotide Trp2 epitope-encoding nucleic acid sequence. In exampleswhere repeats are separated by 2, 3, 4, 5, 6, 7, 8, or 9 separatedistinct epitope-encoding nucleic acid sequence, and eachepitope-encoding nucleic acid sequences (inclusive of optional 5′ linkersequence and/or the optional 3′ linker sequences) encodes a peptide 25amino acids in length, the repeats can be separated by 150, 225, 300,375, 450, 525, 600, or 675 nucleotides, respectively.

In one embodiment, different peptides and/or polypeptides or nucleicacid sequences encoding them are selected so that the peptides and/orpolypeptides capable of associating with different MHC molecules, suchas different MHC class I molecules and/or different MHC class IImolecules. In some aspects, one vaccine composition comprises codingsequence for peptides and/or polypeptides capable of associating withthe most frequently occurring MHC class I molecules and/or different MHCclass II molecules. Hence, vaccine compositions can comprise differentfragments capable of associating with at least 2 preferred, at least 3preferred, or at least 4 preferred MHC class I molecules and/ordifferent MHC class II molecules.

The vaccine composition can be capable of raising a specific cytotoxicT-cells response and/or a specific helper T-cell response.

A vaccine composition can further comprise an adjuvant and/or a carrier.Examples of useful adjuvants and carriers are given herein below. Acomposition can be associated with a carrier such as e.g. a protein oran antigen-presenting cell such as e.g. a dendritic cell (DC) capable ofpresenting the peptide to a T-cell.

Adjuvants are any substance whose admixture into a vaccine compositionincreases or otherwise modifies the immune response to an antigen.Carriers can be scaffold structures, for example a polypeptide or apolysaccharide, to which an antigen, is capable of being associated.Optionally, adjuvants are conjugated covalently or non-covalently.

The ability of an adjuvant to increase an immune response to an antigenis typically manifested by a significant or substantial increase in animmune-mediated reaction, or reduction in disease symptoms. For example,an increase in humoral immunity is typically manifested by a significantincrease in the titer of antibodies raised to the antigen, and anincrease in T-cell activity is typically manifested in increased cellproliferation, or cellular cytotoxicity, or cytokine secretion. Anadjuvant may also alter an immune response, for example, by changing aprimarily humoral or Th response into a primarily cellular, or Thresponse.

Suitable adjuvants include, but are not limited to 1018 ISS, alum,aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM,GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, MontanideIMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,OK-432, OM-174, OM-197-MP-E_(C), ONTAK, PepTel vector system, PLGmicroparticles, resiquimod, SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived fromsaponin, mycobacterial extracts and synthetic bacterial cell wallmimics, and other proprietary adjuvants such as Ribi's Detox. Quil orSuperfos. Adjuvants such as incomplete Freund's or GM-CSF are useful.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Dupuis M, etal., Cell Immunol. 1998; 186(1):18-27; Allison A C; Dev Biol Stand.1998; 92:3-11). Also cytokines can be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-alpha), accelerating the maturation of dendriticcells into efficient antigen-presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specificallyincorporated herein by reference in its entirety) and acting asimmunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J ImmunotherEmphasis Tumor Immunol. 1996 (6):414-418).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Other TLR bindingmolecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also beused.

Other examples of useful adjuvants include, but are not limited to,chemically modified CpGs (e.g. CpR, Idera), Poly(I:C)(e.g. polyi:CI2U),non-CpG bacterial DNA or RNA as well as immunoactive small molecules andantibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex,NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999,CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives can readily bedetermined by the skilled artisan without undue experimentation.Additional adjuvants include colony-stimulating factors, such asGranulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).

A vaccine composition can comprise more than one different adjuvant.Furthermore, a therapeutic composition can comprise any adjuvantsubstance including any of the above or combinations thereof. It is alsocontemplated that a vaccine and an adjuvant can be administered togetheror separately in any appropriate sequence.

A carrier (or excipient) can be present independently of an adjuvant.The function of a carrier can for example be to increase the molecularweight of in particular mutant to increase activity or immunogenicity,to confer stability, to increase the biological activity, or to increaseserum half-life. Furthermore, a carrier can aid presenting peptides toT-cells. A carrier can be any suitable carrier known to the personskilled in the art, for example a protein or an antigen presenting cell.A carrier protein could be but is not limited to keyhole limpethemocyanin, serum proteins such as transferrin, bovine serum albumin,human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, orhormones, such as insulin or palmitic acid. For immunization of humans,the carrier is generally a physiologically acceptable carrier acceptableto humans and safe. However, tetanus toxoid and/or diptheria toxoid aresuitable carriers. Alternatively, the carrier can be dextrans forexample sepharose.

Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptidebound to an MHC molecule rather than the intact foreign antigen itself.The MHC molecule itself is located at the cell surface of an antigenpresenting cell. Thus, an activation of CTLs is possible if a trimericcomplex of peptide antigen, MHC molecule, and APC is present.Correspondingly, it may enhance the immune response if not only thepeptide is used for activation of CTLs, but if additionally APCs withthe respective MHC molecule are added. Therefore, in some embodiments avaccine composition additionally contains at least one antigenpresenting cell.

Antigens can also be included in viral vector-based vaccine platforms,such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus,adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy(2004) 10, 616-629), or lentivirus, including but not limited to second,third or hybrid second/third generation lentivirus and recombinantlentivirus of any generation designed to target specific cell types orreceptors (See, e.g., Hu et al., Immunization Delivered by LentiviralVectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61, Sakuma et al., Lentiviral vectors: basic to translational,Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue ofsplicing-mediated intron loss maximizes expression in lentiviral vectorscontaining the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector forSafe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12):9873-9880). Dependent on the packaging capacity of the above mentionedviral vector-based vaccine platforms, this approach can deliver one ormore nucleic acid sequences that encode one or more antigenic peptides.The sequences may be flanked by non-mutated sequences, may be separatedby linkers or may be preceded with one or more sequences targeting asubcellular compartment (See, e.g., Gros et al., Prospectiveidentification of neoantigen-specific lymphocytes in the peripheralblood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen etal., Targeting of cancer neoantigens with donor-derived T cell receptorrepertoires, Science. (2016) 352 (6291):1337-41, Lu et al., Efficientidentification of mutated cancer antigens recognized by T cellsassociated with durable tumor regressions, Clin Cancer Res. (2014)20(13):3401-10). Upon introduction into a host, infected cells expressthe antigens, and thereby elicit a host immune (e.g., CTL) responseagainst the peptide(s). Vaccinia vectors and methods useful inimmunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.Another vector is BCG (Bacille Calmette Guerin). BCG vectors aredescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vaccine vectors useful for therapeutic administration orimmunization of antigens, e.g., Salmonella typhi vectors, and the likewill be apparent to those skilled in the art from the descriptionherein.

Also disclosed herein is an adenoviral vector delivery compositioncapable of delivering one or more payload nucleic acid sequences. Apayload nucleic acid sequence can be any nucleic acid sequence desiredto be delivered to a cell of interest. In general, the payload is anucleic acid sequence linked to a promoter to drive expression of thenucleic acid sequence. The payload nucleic acid sequence can encode apolypeptide (i.e., a nucleic acid sequence capable of being transcribedand translated into a protein). In general, a payload nucleic acidsequence encoding a peptide can encode any protein desired to beexpressed in a cell. Examples of proteins include, but are not limitedto, an antigen (e.g., a MHC class I epitope, a MHC class II epitope, oran epitope capable of stimulating a B cell response), an antibody, acytokine, a chimeric antigen receptor (CAR), a T-cell receptor, or agenome-editing system component (e.g., a nuclease used in agenome-editing system). Genome-editing systems include, but are notlimited to, a CRISPR system, a zinc-finger system, a meganucleasesystem, or a TALEN system. The payload nucleic acid sequence can benon-coding (i.e., a nucleic acid sequence capable of being transcribedbut is not translated into a protein). In general, a non-coding payloadnucleic acid sequence can be any non-coding polynucleotide desired to beexpressed in a cell. Examples of non-coding polynucleotides include, butare not limited to, RNA interference (RNAi) polynucleotides (e.g.,antisense oligonucleotides, shRNAs, siRNAs, miRNAs etc.) orgenome-editing system polynucleotide (e.g., a guide RNA [gRNA], asingle-guide RNA [sgRNA], a trans-activating CRISPR [tracrRNA], and/or aCRISPR RNA [crRNA]). A payload nucleic acid sequence can encode two ormore (e.g., 2, 3, 4, 5 or more) distinct polypeptides (e.g., two or moredistinct epitope sequences linked together) or contain two or moredistinct non-coding nucleic acid sequences (e.g., two or more distinctRNAi polynucleotides). A payload nucleic acid sequence can have acombination of polypeptide-encoding nucleic acid sequences andnon-coding nucleic acid sequences.

V.A.1 Cassettes

The methods employed for the selection of one or more antigens, thecloning and construction of a “cassette” and its insertion into a viralvector are within the skill in the art given the teachings providedherein. A cassette can have one or more payload nucleic acid sequences,such as a cassette containing multiple payload nucleic acid sequenceseach independently operably linked to separate promoters and/or linkedtogether using other multicistonic systems, such as 2A ribosome skippingsequence elements (e.g., E2A, P2A, F2A, or T2A sequences) or InternalRibosome Entry Site (IRES) sequence elements. In a cassette containingmore than one payload nucleic acid sequence, each payload nucleic acidsequence can contain one or more payloads, e.g., each payload nucleicacid sequence can encode two or more polypeptides or contain two or morenon-coding nucleic acid sequences. A cassette can have a combination ofpolypeptide-encoding nucleic acid sequences and non-coding nucleic acidsequences.

A cassette can be an antigen cassette. By “antigen cassette” is meantthe combination of a selected antigen or plurality of antigens and theother regulatory elements necessary to transcribe the antigen(s) andexpress the transcribed product. Antigen cassettes can include one ormore antigens. The selected antigen or plurality of antigens can referto distinct epitope sequences, e.g., an antigen-encoding nucleic acidsequence in the cassette can encode an epitope-encoding nucleic acidsequence (or plurality of epitope-encoding nucleic acid sequences) suchthat the epitopes are transcribed and expressed.

A payload nucleic acid sequence or plurality of payload nucleic acidsequences can be operatively linked to regulatory components in a mannerwhich permits transcription. Such components include conventionalregulatory elements that can drive expression of the antigen(s) in acell transfected with the viral vector. Thus the cassette can alsocontain a selected promoter which is linked to the payload nucleic acidsequence(s) and located, with other, optional regulatory elements,within the selected viral sequences of the recombinant vector.

Useful promoters can be constitutive promoters or regulated (e.g.,inducible) promoters, which will enable control of the amount of payloadnucleic acid sequence(s), and in general the amount of a peptide (e.g.,an antigen) in the case of coding payload nucleic acid sequences, to beexpressed. For example, a desirable promoter is that of thecytomegalovirus immediate early promoter/enhancer [see, e.g., Boshart etal, Cell, 41:521-530 (1985)]. Another desirable promoter includes theRous sarcoma virus LTR promoter/enhancer. Still anotherpromoter/enhancer sequence is the chicken cytoplasmic beta-actinpromoter [T. A. Kost et al, Nucl. Acids Res., 11(23):8287 (1983)]. Othersuitable or desirable promoters can be selected by one of skill in theart, such as a CMV, SV40, EF-1, RSV, PGK, HSA, MCK or EBV promotersequence.

The cassette can also include nucleic acid sequences heterologous to theviral vector sequences including sequences providing signals forefficient polyadenylation of the transcript (poly(A), poly-A or pA) andintrons with functional splice donor and acceptor sites. A common poly-Asequence which is employed in the exemplary vectors of this invention isthat derived from the papovavirus SV-40. The poly-A sequence generallycan be inserted in the cassette following the payload nucleic acidsequences and before the viral vector sequences. A common intronsequence can also be derived from SV-40, and is referred to as the SV-40T intron sequence. A cassette can also contain such an intron, locatedbetween the promoter/enhancer sequence and the payload nucleic acidsequence(s). Selection of these and other common vector elements areconventional [see, e.g., Sambrook et al, “Molecular Cloning. ALaboratory Manual.”, 2d edit., Cold Spring Harbor Laboratory, New York(1989) and references cited therein] and many such sequences areavailable from commercial and industrial sources as well as fromGenbank.

A cassette can have one or more payload nucleic acid sequences. Forexample, a given cassette can include 1-10, 1-20, 1-30, 10-20, 15-25,15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more payload nucleic acid sequences. Payload nucleic acidsequences can be linked directly to one another. Payload nucleic acidsequences can also be linked to one another with linkers.

A cassette can have one or more payload nucleic acid sequences encodinga polypeptide. For example, a given cassette can include 1-10, 1-20,1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more payload nucleic acid sequencesencoding a polypeptide. A cassette can have one or more payload nucleicacid sequences where each payload nucleic acid sequence encodes adistinct polypeptide. A cassette can have one or more payload nucleicacid sequences where each payload nucleic acid sequence encodes one ormore polypeptides. A cassette can have one or more payload nucleic acidsequences where one or more payload nucleic acid sequences encode one ormore polypeptides. Polypeptides encoded by a payload nucleic acidsequence can be in any orientation relative to one another including Nto C or C to N.

An antigen cassette can have one or more antigens. For example, a givencassette can include 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or moreantigens. Antigens can be linked directly to one another. Antigens canalso be linked to one another with linkers. Antigens can be in anyorientation relative to one another including N to C or C to N.

As above stated, the cassette can be located in the site of any selecteddeletion in the viral vector, such as the site of the E1 gene regiondeletion or E3 gene region deletion, among others which may be selected.

The cassette can be described using the following formula to describethe ordered sequence of each element, from 5′ to 3′:

(P_(a)-(L5_(b)-N_(c)-L3_(d))_(X))_(Z)-(P2_(h)-(G5_(e)-U_(f))_(Y))_(W)-G3_(g)

wherein P and P2 comprise promoter nucleic acid sequences, N comprises adistinct epitope-encoding nucleic acid sequence, L5 comprises a 5′linker sequence, L3 comprises a 3′ linker sequence, G5 comprises anucleic acid sequences encoding an amino acid linker, G3 comprises oneof the at least one nucleic acid sequences encoding an amino acidlinker, U comprises an MHC class II antigen-encoding nucleic acidsequence, where for each X the corresponding Nc is a epitope encodingnucleic acid sequence, where for each Y the corresponding Uf is anantigen-encoding nucleic acid sequence. The composition and orderedsequence can be further defined by selecting the number of elementspresent, for example where a=0 or 1, where b=0 or 1, where c=1, whered=0 or 1, where e=0 or 1, where f=1, where g=0 or 1, where h=0 or 1, X=1to 400, Y=0, 1, 2, 3, 4 or 5, Z=1 to 400, and W=0, 1, 2, 3, 4 or 5.

In one example, elements present include where a=0, b=1, d=1, e=1, g=1,h=0, X=10, Y=2, Z=1, and W=1, describing where no additional promoter ispresent (i.e. only the promoter nucleic acid sequence provided by theRNA alphavirus backbone is present), 20 MHC class I epitope are present,a 5′ linker is present for each N, a 3′ linker is present for each N, 2MHC class II epitopes are present, a linker is present linking the twoMHC class II epitopes, a linker is present linking the 5′ end of the twoMHC class II epitopes to the 3′ linker of the final MHC class I epitope,and a linker is present linking the 3′ end of the two MHC class IIepitopes to the to the RNA alphavirus backbone. Examples of linking the3′ end of the cassette to the RNA alphavirus backbone include linkingdirectly to the 3′ UTR elements provided by the RNA alphavirus backbone,such as a 3′ 19-nt CSE. Examples of linking the 5′ end of the cassetteto the RNA alphavirus backbone include linking directly to a 26Spromoter sequence, an alphavirus 5′ UTR, a 51-nt CSE, or a 24-nt CSE.

Other examples include: where a=1 describing where a promoter other thanthe promoter nucleic acid sequence provided by the RNA alphavirusbackbone is present; where a=1 and Z is greater than 1 where multiplepromoters other than the promoter nucleic acid sequence provided by theRNA alphavirus backbone are present each driving expression of 1 or moredistinct MHC class I epitope encoding nucleic acid sequences; where h=1describing where a separate promoter is present to drive expression ofthe MHC class II antigen-encoding nucleic acid sequences; and where g=0describing the MHC class II antigen-encoding nucleic acid sequence, ifpresent, is directly linked to the RNA alphavirus backbone.

Other examples include where each MHC class I epitope that is presentcan have a 5′ linker, a 3′ linker, neither, or both. In examples wheremore than one MHC class I epitope is present in the same cassette, someMHC class I epitopes may have both a 5′ linker and a 3′ linker, whileother MHC class I epitopes may have either a 5′ linker, a 3′ linker, orneither. In other examples where more than one MHC class I epitope ispresent in the same cassette, some MHC class I epitopes may have eithera 5′ linker or a 3′ linker, while other MHC class I epitopes may haveeither a 5′ linker, a 3′ linker, or neither.

In examples where more than one MHC class II epitope is present in thesame cassette, some MHC class II epitopes may have both a 5′ linker anda 3′ linker, while other MHC class II epitopes may have either a 5′linker, a 3′ linker, or neither. In other examples where more than oneMHC class II epitope is present in the same cassette, some MHC class IIepitopes may have either a 5′ linker or a 3′ linker, while other MHCclass II epitopes may have either a 5′ linker, a 3′ linker, or neither.

The promoter nucleic acid sequences P and/or P2 can be the same as apromoter nucleic acid sequence provided by the RNA alphavirus backbone.For example, the promoter sequence provided by the RNA alphavirusbackbone, Pn and P2, can each comprise a 26S subgenomic promoter. Thepromoter nucleic acid sequences P and/or P2 can be different from thepromoter nucleic acid sequence provided by the RNA alphavirus backbone,as well as can be different from each other.

The 5′ linker L5 can be a native sequence or a non-natural sequence.Non-natural sequence include, but are not limited to, AAY, RR, and DPP.The 3′ linker L3 can also be a native sequence or a non-naturalsequence. Additionally, L5 and L3 can both be native sequences, both benon-natural sequences, or one can be native and the other non-natural.For each X, the amino acid linkers can be 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or more amino acids in length. For each X, the amino acid linkers can bealso be at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, orat least 30 amino acids in length.

The amino acid linker G5, for each Y, can be 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or more amino acids in length. For each Y, the amino acid linkers can bealso be at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, orat least 30 amino acids in length.

The amino acid linker G3 can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or moreamino acids in length. G3 can be also be at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, or at least 30 amino acids in length.

For each X, each N can encodes a MHC class I epitope 7-15 amino acids inlength. For each X, each N can also encodes a MHC class I epitope 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acids in length. For each X, each N can alsoencodes a MHC class I epitope at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, orat least 30 amino acids in length.

The cassette encoding the payload nucleic acid sequence can be 700nucleotides or less. The cassette encoding the payload nucleic acidsequence can be 700 nucleotides or less and encode 2 distinctepitope-encoding nucleic acid sequences. The cassette encoding thepayload nucleic acid sequence can be 700 nucleotides or less and encodeat least 2 distinct epitope-encoding nucleic acid sequences. Thecassette encoding the payload nucleic acid sequence can be 700nucleotides or less and encode 3 distinct epitope-encoding nucleic acidsequences. The cassette encoding the payload nucleic acid sequence canbe 700 nucleotides or less and encode at least 3 distinctepitope-encoding nucleic acid sequences. The cassette encoding thepayload nucleic acid sequence can be 700 nucleotides or less and include1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

The cassette encoding the payload nucleic acid sequence can be between375-700 nucleotides in length. The cassette encoding the payload nucleicacid sequence can be between 375-700 nucleotides in length and encode 2distinct epitope-encoding nucleic acid sequences. The cassette encodingthe payload nucleic acid sequence can be between 375-700 nucleotides inlength and encode at least 2 distinct epitope-encoding nucleic acidsequences. The cassette encoding the payload nucleic acid sequence canbe between 375-700 nucleotides in length and encode 3 distinctepitope-encoding nucleic acid sequences. The cassette encoding thepayload nucleic acid sequence be between 375-700 nucleotides in lengthand encode at least 3 distinct epitope-encoding nucleic acid sequences.The cassette encoding the payload nucleic acid sequence can be between375-700 nucleotides in length and include 1-10, 1-5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more antigens.

The cassette encoding the payload nucleic acid sequence can be 600, 500,400, 300, 200, or 100 nucleotides in length or less. The cassetteencoding the payload nucleic acid sequence can be 600, 500, 400, 300,200, or 100 nucleotides in length or less and encode 2 distinctepitope-encoding nucleic acid sequences. The cassette encoding thepayload nucleic acid sequence can be 600, 500, 400, 300, 200, or 100nucleotides in length or less and encode at least 2 distinctepitope-encoding nucleic acid sequences. The cassette encoding thepayload nucleic acid sequence can be 600, 500, 400, 300, 200, or 100nucleotides in length or less and encode 3 distinct epitope-encodingnucleic acid sequences. The cassette encoding the payload nucleic acidsequence can be 600, 500, 400, 300, 200, or 100 nucleotides in length orless and encode at least 3 distinct epitope-encoding nucleic acidsequences. The cassette encoding the payload nucleic acid sequence canbe 600, 500, 400, 300, 200, or 100 nucleotides in length or less andinclude 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

The cassette encoding the payload nucleic acid sequence can be between375-600, between 375-500, or between 375-400 nucleotides in length. Thecassette encoding the payload nucleic acid sequence can be between375-600, between 375-500, or between 375-400 nucleotides in length andencode 2 distinct epitope-encoding nucleic acid sequences. The cassetteencoding the payload nucleic acid sequence can be between 375-600,between 375-500, or between 375-400 nucleotides in length and encode atleast 2 distinct epitope-encoding nucleic acid sequences. The cassetteencoding the payload nucleic acid sequence can be between 375-600,between 375-500, or between 375-400 nucleotides in length and encode 3distinct epitope-encoding nucleic acid sequences. The cassette encodingthe payload nucleic acid sequence can be between 375-600, between375-500, or between 375-400 nucleotides in length and encode at least 3distinct epitope-encoding nucleic acid sequences. The cassette encodingthe payload nucleic acid sequence can be between 375-600, between375-500, or between 375-400 nucleotides in length and include 1-10, 1-5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

V.A.2 TET Promoter System

Also disclosed herein is a viral vector comprising a cassette with atleast one payload sequence operably linked to a regulatable promoterthat is a TET promoter system, such as a TET-On system or TET-Offsystem. Without wishing to be bound by theory, a TET promoter system canbe used to minimize transcription of payload nucleic acids encoded in acassette, such as antigens encoded in a vaccine cassette, during viralproduction. A TET promoter system can include a tetracycline (TET)repressor protein (TETr) controlled promoter. Accordingly, alsodisclosed herein is a viral vector comprising a cassette with at leastone payload sequence operably linked to a tetracycline (TET) repressorprotein (TETr) controlled promoter. TETr sequences (tTS) can include theamino acid sequence shown in a SEQ ID NO:63 and/or encoded by thenucleotide sequence shown in SEQ ID NO:62. A TETr controlled promotercan include the 19 bp TET operator (TETo) sequence TCCCTATCAGTGATAGAGA(SEQ ID NO:60). A TETr controlled promoter can include 2, 3, 4, 5, 6, 7,8, 9, or 10 or more TETo nucleic acid sequences. In TETr controlledpromoter have 2 or more TETo nucleic acid sequences, the TETo sequencescan be linked together. In TETr controlled promoter have 2 or more TETonucleic acid sequences, the TETo sequences can be directly linkedtogether. In TETr controlled promoter have 2 or more TETo nucleic acidsequences, the TETo sequences can be linked together with a linkersequence, such as a linker sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides. Inone example, the linker sequence has the linker nucleotide sequenceshown in SEQ ID NO:61. In general, a TETr controlled promoter can useany promoter sequence desired, such as a SV40, EF-1, RSV, PGK, HSA, MCKor EBV promoter sequence. A TETr controlled promoter can use a CMVpromoter sequence. A TETr controlled promoter can use a minimal CMVpromoter sequence. TETo sequences can be upstream (5′) of a promotersequence region where RNA polymerase binds. In an illustrative example,7 TETo sequences are upstream (5′) of a promoter sequence. A TETrcontrolled promoter operably linked to the at least one payload nucleicacid sequence with TETo sequence upstream of the promoter sequenceregion can have an ordered sequence described in the formula, from 5′ to3′:

(T-L_(Y))_(X)-P—N

where N is a payload nucleic acid sequence, P is a RNA polymerasebinding sequence of the promoter sequence operably linked to payloadnucleic acid sequence, T is a TETo nucleic acid sequences comprising thenucleotide sequence shown in SEQ ID NO:60, L is a linker sequence, whereY=0 or 1 for each X, and wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20. In an illustrative example, X=7and Y=1 for each X describes where 7 TETo sequences are upstream (5′) ofthe promoter sequence and each TETo sequence is separated by a linker.

A TETo sequences can be downstream (3′) of a promoter sequence regionwhere RNA polymerase binds. In another illustrative example, 2 TETosequences are downstream (3′) of a promoter sequence. A TETr controlledpromoter operably linked to the at least one payload nucleic acidsequence with TETo sequence downstream of the promoter sequence regioncan have an ordered sequence described in the formula, from 5′ to 3′:

P-(T-L_(Y))_(X)-N

where N is a payload nucleic acid sequence, P is a RNA polymerasebinding sequence of the promoter sequence operably linked to payloadnucleic acid sequence, T is a TETo nucleic acid sequences comprising thenucleotide sequence shown in SEQ ID NO:60, L is a linker sequence, whereY=0 or 1 for each X, and wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20. In an illustrative example, X=2and Y=1 for each X describes where 2 TETo sequences are downstream (3′)of the promoter sequence and each TETo sequence is separated by alinker.

Viral production of vectors with TETr controlled promoters can use anyviral production cell line engineered to express a TETr sequence (tTS),such as a 293 cell line or its derivatives (e.g., a 293F cell line)engineered to express tTS. Viral production of vectors with TETrcontrolled promoters in tTS-expressing cell can improve viralproduction. Viral production of vectors with TETr controlled promotersin tTS-expressing cell can improve viral infectivity defined as viralparticles (VP) per infectious unit (IU). Viral production of vectorswith TETr controlled promoters in tTS-expressing cell can improve viralproduction and/or viral infectivity by at least 1.5, at least 2, atleast 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least5, at least 6, at least 7, at least 8, at least 9, or at least 10-foldrelative to production in a non-tTS-expressing cell. Viral production ofvectors with TETr controlled promoters in tTS-expressing cell canimprove viral production and/or viral infectivity by at least 15, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 60, at least 70, at least 80, at least 90, orat least 100-fold relative to production in a non-tTS-expressing cell.Viral production of vectors with TETr controlled promoters intTS-expressing cell can improve viral production and/or viralinfectivity by at least 1.5, at least 2, at least 2.5, at least 3, atleast 3.5, at least 4, at least 4.5, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10-fold relative to production of avector not having a TETr controlled promoter. Viral production ofvectors with TETr controlled promoters in tTS-expressing cell canimprove viral production and/or viral infectivity by at least 15, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 60, at least 70, at least 80, at least 90, orat least 100-fold relative to production of a vector not having a TETrcontrolled promoter.

V.B. Immune Checkpoints

Vectors described herein, such as C68 vectors described herein oralphavirus vectors described herein, can comprise a nucleic acid whichencodes at least one antigen and the same or a separate vector cancomprise a nucleic acid which encodes at least one immune modulator(e.g., an antibody such as an scFv) which binds to and blocks theactivity of an immune checkpoint molecule. Vectors can comprise acassette and one or more nucleic acid molecules encoding a checkpointinhibitor.

Illustrative immune checkpoint molecules that can be targeted forblocking or inhibition include, but are not limited to, CTLA-4, 4-1BB(CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM,TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2family of molecules and is expressed on all NK, □□, and memory CD8+ (□□)T cells), CD160 (also referred to as BY55), and CGEN-15049. Immunecheckpoint inhibitors include antibodies, or antigen binding fragmentsthereof, or other binding proteins, that bind to and block or inhibitthe activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4,BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160,and CGEN-15049. Illustrative immune checkpoint inhibitors includeTremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonalAntibody (Anti-B7-H1; MEDI4736), ipilimumab, MK-3475 (PD-1 blocker),Nivolumamb (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1antibody) and Yervoy/ipilimumab (anti-CTLA-4 checkpoint inhibitor).Antibody-encoding sequences can be engineered into vectors such as C68using ordinary skill in the art. An exemplary method is described inFang et al., Stable antibody expression at therapeutic levels using the2A peptide. Nat Biotechnol. 2005 May; 23(5):584-90. Epub 2005 Apr. 17;herein incorporated by reference for all purposes.

V.C. Additional Considerations for Vaccine Design and Manufacture

V.C.1. Determination of a Set of Peptides that Cover all Tumor Subclones

Truncal peptides, meaning those presented by all or most tumorsubclones, can be prioritized for inclusion into the vaccine.⁵³Optionally, if there are no truncal peptides predicted to be presentedand immunogenic with high probability, or if the number of truncalpeptides predicted to be presented and immunogenic with high probabilityis small enough that additional non-truncal peptides can be included inthe vaccine, then further peptides can be prioritized by estimating thenumber and identity of tumor subclones and choosing peptides so as tomaximize the number of tumor subclones covered by the vaccine.⁵⁴

V.C.2. Antigen Prioritization

After all of the above antigen filters are applied, more candidateantigens may still be available for vaccine inclusion than the vaccinetechnology can support. Additionally, uncertainty about various aspectsof the antigen analysis may remain and tradeoffs may exist betweendifferent properties of candidate vaccine antigens. Thus, in place ofpredetermined filters at each step of the selection process, anintegrated multi-dimensional model can be considered that placescandidate antigens in a space with at least the following axes andoptimizes selection using an integrative approach.

-   -   1. Risk of auto-immunity or tolerance (risk of germline) (lower        risk of auto-immunity is typically preferred)    -   2. Probability of sequencing artifact (lower probability of        artifact is typically preferred)    -   3. Probability of immunogenicity (higher probability of        immunogenicity is typically preferred)    -   4. Probability of presentation (higher probability of        presentation is typically preferred)    -   5. Gene expression (higher expression is typically preferred)    -   6. Coverage of HLA genes (larger number of HLA molecules        involved in the presentation of a set of antigens may lower the        probability that a tumor will escape immune attack via        downregulation or mutation of HLA molecules)    -   7. Coverage of HLA classes (covering both HLA-I and HLA-II may        increase the probability of therapeutic response and decrease        the probability of tumor escape)

Additionally, optionally, antigens can be deprioritized (e.g., excluded)from the vaccination if they are predicted to be presented by HLAalleles lost or inactivated in either all or part of the patient's tumoror infected cell. HLA allele loss can occur by either somatic mutation,loss of heterozygosity, or homozygous deletion of the locus. Methods fordetection of HLA allele somatic mutation are well known in the art, e.g.(Shukla et al., 2015). Methods for detection of somatic LOH andhomozygous deletion (including for HLA locus) are likewise welldescribed. (Carter et al., 2012; McGranahan et al., 2017; Van Loo etal., 2010). Antigens can also be deprioritized if mass-spectrometry dataindicates a predicted antigen is not presented by a predicted HLAallele.

V.D. Alphavirus

V.D.1. Alphavirus Biology

Alphaviruses are members of the family Togaviridae, and arepositive-sense single stranded RNA viruses. Members are typicallyclassified as either Old World, such as Sindbis, Ross River, Mayaro,Chikungunya, and Semliki Forest viruses, or New World, such as easternequine encephalitis, Aura, Fort Morgan, or Venezuelan equineencephalitis virus and its derivative strain TC-83 (Strauss MicrobrialReview 1994). A natural alphavirus genome is typically around 12 kb inlength, the first two-thirds of which contain genes encodingnon-structural proteins (nsPs) that form RNA replication complexes forself-replication of the viral genome, and the last third of whichcontains a subgenomic expression cassette encoding structural proteinsfor virion production (Frolov RNA 2001).

A model lifecycle of an alphavirus involves several distinct steps(Strauss Microbrial Review 1994, Jose Future Microbiol 2009). Followingvirus attachment to a host cell, the virion fuses with membranes withinendocytic compartments resulting in the eventual release of genomic RNAinto the cytosol. The genomic RNA, which is in a plus-strand orientationand comprises a 5′ methylguanylate cap and 3′ polyA tail, is translatedto produce non-structural proteins nsP1-4 that form the replicationcomplex. Early in infection, the plus-strand is then replicated by thecomplex into a minus-stand template. In the current model, thereplication complex is further processed as infection progresses, withthe resulting processed complex switching to transcription of theminus-strand into both full-length positive-strand genomic RNA, as wellas the 26S subgenomic positive-strand RNA containing the structuralgenes. Several conserved sequence elements (CSEs) of alphavirus havebeen identified to potentially play a role in the various RNAreplication steps including; a complement of the 5′ UTR in thereplication of plus-strand RNAs from a minus-strand template, a 51-ntCSE in the replication of minus-strand synthesis from the genomictemplate, a 24-nt CSE in the junction region between the nsPs and the26S RNA in the transcription of the subgenomic RNA from theminus-strand, and a 3′ 19-nt CSE in minus-strand synthesis from theplus-strand template.

Following the replication of the various RNA species, virus particlesare then typically assembled in the natural lifecycle of the virus. The26S RNA is translated and the resulting proteins further processed toproduce the structural proteins including capsid protein, glycoproteinsE1 and E2, and two small polypeptides E3 and 6K (Strauss 1994).Encapsidation of viral RNA occurs, with capsid proteins normallyspecific for only genomic RNA being packaged, followed by virionassembly and budding at the membrane surface.

V.D.2. Alphavirus as a Delivery Vector

Alphaviruses (including alphavirus sequences, features, and otherelements) can be used to generate alphavirus-based delivery vectors(also be referred to as alphavirus vectors, alphavirus viral vectors,alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, orself-amplifying RNA (samRNA) vectors). Alphaviruses have previously beenengineered for use as expression vector systems (Pushko 1997, Rheme2004). Alphaviruses offer several advantages, particularly in a vaccinesetting where heterologous antigen expression can be desired. Due to itsability to self-replicate in the host cytosol, alphavirus vectors aregenerally able to produce high copy numbers of the expression cassettewithin a cell resulting in a high level of heterologous antigenproduction. Additionally, the vectors are generally transient, resultingin improved biosafety as well as reduced induction of immunologicaltolerance to the vector. The public, in general, also lacks pre-existingimmunity to alphavirus vectors as compared to other standard viralvectors, such as human adenovirus. Alphavirus based vectors alsogenerally result in cytotoxic responses to infected cells. Cytotoxicity,to a certain degree, can be important in a vaccine setting to properlyillicit an immune response to the heterologous antigen expressed.However, the degree of desired cytotoxicity can be a balancing act, andthus several attenuated alphaviruses have been developed, including theTC-83 strain of VEE. Thus, an example of an antigen expression vectordescribed herein can utilize an alphavirus backbone that allows for ahigh level of antigen expression, elicits a robust immune response toantigen, does not elicit an immune response to the vector itself, andcan be used in a safe manner. Furthermore, the antigen expressioncassette can be designed to elicit different levels of an immuneresponse through optimization of which alphavirus sequences the vectoruses, including, but not limited to, sequences derived from VEE or itsattenuated derivative TC-83.

Several expression vector design strategies have been engineered usingalphavirus sequences (Pushko 1997). In one strategy, a alphavirus vectordesign includes inserting a second copy of the 26S promoter sequenceelements downstream of the structural protein genes, followed by aheterologous gene (Frolov 1993). Thus, in addition to the naturalnon-structural and structural proteins, an additional subgenomic RNA isproduced that expresses the heterologous protein. In this system, allthe elements for production of infectious virions are present and,therefore, repeated rounds of infection of the expression vector innon-infected cells can occur.

Another expression vector design makes use of helper virus systems(Pushko 1997). In this strategy, the structural proteins are replaced bya heterologous gene. Thus, following self-replication of viral RNAmediated by still intact non-structural genes, the 26S subgenomic RNAprovides for expression of the heterologous protein. Traditionally,additional vectors that expresses the structural proteins are thensupplied in trans, such as by co-transfection of a cell line, to produceinfectious virus. A system is described in detail in U.S. Pat. No.8,093,021, which is herein incorporated by reference in its entirety,for all purposes. The helper vector system provides the benefit oflimiting the possibility of forming infectious particles and, therefore,improves biosafety. In addition, the helper vector system reduces thetotal vector length, potentially improving the replication andexpression efficiency. Thus, an example of an antigen expression vectordescribed herein can utilize an alphavirus backbone wherein thestructural proteins are replaced by a cassette, the resulting vectorboth reducing biosafety concerns, while at the same time promotingefficient expression due to the reduction in overall expression vectorsize.

V.D.3. Alphavirus Production In Vitro

Alphavirus delivery vectors are generally positive-sense RNApolynucleotides. A convenient technique well-known in the art for RNAproduction is in vitro transcription IVT. In this technique, a DNAtemplate of the desired vector is first produced by techniqueswell-known to those in the art, including standard molecular biologytechniques such as cloning, restriction digestion, ligation, genesynthesis, and polymerase chain reaction (PCR). The DNA templatecontains a RNA polymerase promoter at the 5′ end of the sequence desiredto be transcribed into RNA. Promoters include, but are not limited to,bacteriophage polymerase promoters such as T3, T7, or SP6. The DNAtemplate is then incubated with the appropriate RNA polymerase enzyme,buffer agents, and nucleotides (NTPs). The resulting RNA polynucleotidecan optionally be further modified including, but limited to, additionof a 5′ cap structure such as 7-methylguanosine or a related structure,and optionally modifying the 3′ end to include a polyadenylate (polyA)tail. The RNA can then be purified using techniques well-known in thefield, such as phenol-chloroform extraction.

V.D.4. Delivery Via Lipid Nanoparticle

An important aspect to consider in vaccine vector design is immunityagainst the vector itself (Riley 2017). This may be in the form ofpreexisting immunity to the vector itself, such as with certain humanadenovirus systems, or in the form of developing immunity to the vectorfollowing administration of the vaccine. The latter is an importantconsideration if multiple administrations of the same vaccine areperformed, such as separate priming and boosting doses, or if the samevaccine vector system is to be used to deliver different cassettes.

In the case of alphavirus vectors, the standard delivery method is thepreviously discussed helper virus system that provides capsid, E1, andE2 proteins in trans to produce infectious viral particles. However, itis important to note that the E1 and E2 proteins are often major targetsof neutralizing antibodies (Strauss 1994). Thus, the efficacy of usingalphavirus vectors to deliver antigens of interest to target cells maybe reduced if infectious particles are targeted by neutralizingantibodies.

An alternative to viral particle mediated gene delivery is the use ofnanomaterials to deliver expression vectors (Riley 2017). Nanomaterialvehicles, importantly, can be made of non-immunogenic materials andgenerally avoid eliciting immunity to the delivery vector itself. Thesematerials can include, but are not limited to, lipids, inorganicnanomaterials, and other polymeric materials. Lipids can be cationic,anionic, or neutral. The materials can be synthetic or naturallyderived, and in some instances biodegradable. Lipids can include fats,cholesterol, phospholipids, lipid conjugates including, but not limitedto, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils,glycerides, and fat soluable vitamins.

Lipid nanoparticles (LNPs) are an attractive delivery system due to theamphiphilic nature of lipids enabling formation of membranes and vesiclelike structures (Riley 2017). In general, these vesicles deliver theexpression vector by absorbing into the membrane of target cells andreleasing nucleic acid into the cytosol. In addition, LNPs can befurther modified or functionalized to facilitate targeting of specificcell types. Another consideration in LNP design is the balance betweentargeting efficiency and cytotoxicity. Lipid compositions generallyinclude defined mixtures of cationic, neutral, anionic, and amphipathiclipids. In some instances, specific lipids are included to prevent LNPaggregation, prevent lipid oxidation, or provide functional chemicalgroups that facilitate attachment of additional moieties. Lipidcomposition can influence overall LNP size and stability. In an example,the lipid composition comprises dilinoleylmethyl-4-dimethylaminobutyrate(MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can beformulated to include one or more other lipids, such as a PEG orPEG-conjugated lipid, a sterol, or neutral lipids.

Nucleic-acid vectors, such as expression vectors, exposed directly toserum can have several undesirable consequences, including degradationof the nucleic acid by serum nucleases or off-target stimulation of theimmune system by the free nucleic acids. Therefore, encapsulation of thealphavirus vector can be used to avoid degradation, while also avoidingpotential off-target affects. In certain examples, an alphavirus vectoris fully encapsulated within the delivery vehicle, such as within theaqueous interior of an LNP. Encapsulation of the alphavirus vectorwithin an LNP can be carried out by techniques well-known to thoseskilled in the art, such as microfluidic mixing and droplet generationcarried out on a microfluidic droplet generating device. Such devicesinclude, but are not limited to, standard T-junction devices orflow-focusing devices. In an example, the desired lipid formulation,such as MC3 or MC3-like containing compositions, is provided to thedroplet generating device in parallel with the alphavirus deliveryvector and other desired agents, such that the delivery vector anddesired agents are fully encapsulated within the interior of the MC3 orMC3-like based LNP. In an example, the droplet generating device cancontrol the size range and size distribution of the LNPs produced. Forexample, the LNP can have a size ranging from 1 to 1000 nanometers indiameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Followingdroplet generation, the delivery vehicles encapsulating the expressionvectors can be further treated or modified to prepare them foradministration.

V.E. Chimpanzee Adenovirus (ChAd)

V.E.1. Viral Delivery with Chimpanzee Adenovirus

Vaccine compositions for delivery of one or more antigens (e.g., via acassette encoding one or more antigens or neoantigens) can be created byproviding adenovirus nucleic acid sequences of chimpanzee origin, avariety of novel vectors, and cell lines expressing chimpanzeeadenovirus genes. A nucleic acid sequence of a chimpanzee C68 adenovirus(also referred to herein as ChAdV68) can be used in a vaccinecomposition for antigen delivery (See SEQ ID NO: 1). Use of C68adenovirus derived vectors is described in further detail in U.S. Pat.No. 6,083,716, which is herein incorporated by reference in itsentirety, for all purposes.

In a further aspect, provided herein is a recombinant adenoviruscomprising the DNA sequence of a chimpanzee adenovirus such as C68 and acassette operatively linked to regulatory sequences directing itsexpression. The recombinant virus is capable of infecting a mammalian,preferably a human, cell and capable of expressing the cassette payloadin the cell. In this vector, the native chimpanzee E1 gene, and/or E3gene, and/or E4 gene can be deleted. A cassette can be inserted into anyof these sites of gene deletion. The cassette can include an antigenagainst which a primed immune response is desired.

In another aspect, provided herein is a mammalian cell infected with achimpanzee adenovirus such as C68.

In still a further aspect, a novel mammalian cell line is provided whichexpresses a chimpanzee adenovirus gene (e.g., from C68) or functionalfragment thereof.

In still a further aspect, provided herein is a method for delivering acassette into a mammalian cell comprising the step of introducing intothe cell an effective amount of a chimpanzee adenovirus, such as C68,that has been engineered to express the cassette.

Still another aspect provides a method for eliciting an immune responsein a mammalian host to treat cancer. The method can comprise the step ofadministering to the host an effective amount of a recombinantchimpanzee adenovirus, such as C68, comprising a cassette that encodesone or more antigens from the tumor against which the immune response istargeted.

Still another aspect provides a method for eliciting an immune responsein a mammalian host to treat or prevent a disease in a subject, such asan infectious disease. The method can comprise the step of administeringto the host an effective amount of a recombinant chimpanzee adenovirus,such as C68, comprising an antigen cassette that encodes one or moreantigens, such as from the infectious disease against which the immuneresponse is targeted.

Also disclosed is a non-simian mammalian cell that expresses achimpanzee adenovirus gene obtained from the sequence of SEQ ID NO: 1.The gene can be selected from the group consisting of the adenovirusE1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 of SEQ ID NO: 1.

Also disclosed is a nucleic acid molecule comprising a chimpanzeeadenovirus DNA sequence comprising a gene obtained from the sequence ofSEQ ID NO: 1. The gene can be selected from the group consisting of saidchimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5genes of SEQ ID NO: 1. In some aspects the nucleic acid moleculecomprises SEQ ID NO: 1. In some aspects the nucleic acid moleculecomprises the sequence of SEQ ID NO: 1, lacking at least one geneselected from the group consisting of E1A, E1B, E2A, E2B, E3, E4, L1,L2, L3, L4 and L5 genes of SEQ ID NO: 1.

Also disclosed is a vector comprising a chimpanzee adenovirus DNAsequence obtained from SEQ ID NO: 1 and a cassette operatively linked toone or more regulatory sequences which direct expression of the cassettein a heterologous host cell, optionally wherein the chimpanzeeadenovirus DNA sequence comprises at least the cis-elements necessaryfor replication and virion encapsidation, the cis-elements flanking thecassette and regulatory sequences. In some aspects, the chimpanzeeadenovirus DNA sequence comprises a gene selected from the groupconsisting of E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genesequences of SEQ ID NO: 1. In some aspects the vector can lack the E1Aand/or E1B gene.

Also disclosed herein is a host cell transfected with a vector disclosedherein such as a C68 vector engineered to expression a cassette. Alsodisclosed herein is a human cell that expresses a selected geneintroduced therein through introduction of a vector disclosed hereininto the cell.

Also disclosed herein is a adenovirus vector comprising: a partiallydeleted E4 gene comprising a deleted or partially-deleted E4orf2 regionand a deleted or partially-deleted E4orf3 region, and optionally adeleted or partially-deleted E4orf4 region. The partially deleted E4 cancomprise an E4 deletion of at least nucleotides 34,916 to 35,642 of thesequence shown in SEQ ID NO:1, and wherein the vector comprises at leastnucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1. Thepartially deleted E4 can comprise an E4 deletion of at least a partialdeletion of nucleotides 34,916 to 34,942 of the sequence shown in SEQ IDNO:1, at least a partial deletion of nucleotides 34,952 to 35,305 of thesequence shown in SEQ ID NO:1, and at least a partial deletion ofnucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO:1, andwherein the vector comprises at least nucleotides 2 to 36,518 of thesequence set forth in SEQ ID NO:1 The partially deleted E4 can comprisean E4 deletion of at least nucleotides 34,980 to 36,516 of the sequenceshown in SEQ ID NO:1, and wherein the vector comprises at leastnucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1. Thepartially deleted E4 can comprise an E4 deletion of at least nucleotides34,979 to 35,642 of the sequence shown in SEQ ID NO:1, and wherein thevector comprises at least nucleotides 2 to 36,518 of the sequence setforth in SEQ ID NO:1. The partially deleted E4 can comprise an E4deletion of at least a partial deletion of E4Orf2, a fully deletedE4Orf3, and at least a partial deletion of E4Orf4. The partially deletedE4 can comprise an E4 deletion of at least a partial deletion of E4Orf2,at least a partial deletion of E4Orf3, and at least a partial deletionof E4Orf4. The partially deleted E4 can comprise an E4 deletion of atleast a partial deletion of E4Orf1, a fully deleted E4Orf2, and at leasta partial deletion of E4Orf3. The partially deleted E4 can comprise anE4 deletion of at least a partial deletion of E4Orf2 and at least apartial deletion of E4Orf3. The partially deleted E4 can comprise an E4deletion between the start site of E4Orf1 to the start site of E4Orf5.The partially deleted E4 can be an E4 deletion adjacent to the startsite of E4Orf1. The partially deleted E4 can be an E4 deletion adjacentto the start site of E4Orf2. The partially deleted E4 can be an E4deletion adjacent to the start site of E4Orf3. The partially deleted E4can be an E4 deletion adjacent to the start site of E4Orf4. The E4deletion can be at least 50, at least 100, at least 200, at least 300,at least 400, at least 500, at least 600, at least 700, at least 800, atleast 900, at least 1000, at least 1100, at least 1200, at least 1300,at least 1400, at least 1500, at least 1600, at least 1700, at least1800, at least 1900, or at least 2000 nucleotides. The E4 deletion canbe at least 700 nucleotides. The E4 deletion can be at least 1500nucleotides. The E4 deletion can be 50 or less, 100 or less, 200 orless, 300 or less, 400 or less, 500 or less, 600 or less, 700 or less,800 or less, 900 or less, 1000 or less, 1100 or less, 1200 or less, 1300or less, 1400 or less, 1500 or less, 1600 or less, 1700 or less, 1800 orless, 1900 or less, or 2000 or less nucleotides. The E4 deletion can be750 nucleotides or less. The E4 deletion can be at least 1550nucleotides or less.

The partially deleted E4 gene can be the E4 gene sequence shown in SEQID NO:1 that lacks at least nucleotides 34,916 to 35,642 of the sequenceshown in SEQ ID NO:1. The partially deleted E4 gene can be the E4 genesequence shown in SEQ ID NO:1 that lacks the E4 gene sequence shown inSEQ ID NO:1 and that lacks at least nucleotides 34,916 to 34,942,nucleotides 34,952 to 35,305 of the sequence shown in SEQ ID NO:1, andnucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO:1. Thepartially deleted E4 gene can be the E4 gene sequence shown in SEQ IDNO:1 and that lacks at least nucleotides 34,980 to 36,516 of thesequence shown in SEQ ID NO:1. The partially deleted E4 gene can be theE4 gene sequence shown in SEQ ID NO:1 and that lacks at leastnucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO:1. Theadenovirus vector having the partially deleted E4 gene can have acassette, wherein the cassette comprises at least one payload nucleicacid sequence, and wherein the cassette comprises at least one promotersequence operably linked to the at least one payload nucleic acidsequence. The adenovirus vector having the partially deleted E4 gene canhave one or more genes or regulatory sequences of the ChAdV68 sequenceshown in SEQ ID NO: 1, optionally wherein the one or more genes orregulatory sequences comprise at least one of the chimpanzee adenovirusinverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3,L4, and L5 genes of the sequence shown in SEQ ID NO: 1. The adenovirusvector having the partially deleted E4 gene can have nucleotides 2 to34,916 of the sequence shown in SEQ ID NO:1, wherein the partiallydeleted E4 gene is 3′ of the nucleotides 2 to 34,916, and optionally thenucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of thesequence shown in SEQ ID NO:1 corresponding to an E1 deletion and/orlack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1corresponding to an E3 deletion. The adenovirus vector having thepartially deleted E4 gene can have nucleotides 35,643 to 36,518 of thesequence shown in SEQ ID NO:1, and wherein the partially deleted E4 geneis 5′ of the nucleotides 35,643 to 36,518. The adenovirus vector havingthe partially deleted E4 gene can have nucleotides 2 to 34,916 of thesequence shown in SEQ ID NO:1, wherein the partially deleted E4 gene is3′ of the nucleotides 2 to 34,916, the nucleotides 2 to 34,916additionally lack nucleotides 577 to 3403 of the sequence shown in SEQID NO:1 corresponding to an E1 deletion and lack nucleotides 27,125 to31,825 of the sequence shown in SEQ ID NO:1 corresponding to an E3deletion. The adenovirus vector having the partially deleted E4 gene canhave nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO:1,wherein the partially deleted E4 gene is 3′ of the nucleotides 2 to34,916, the nucleotides 2 to 34,916 additionally lack nucleotides 577 to3403 of the sequence shown in SEQ ID NO:1 corresponding to an E1deletion and lack nucleotides 27,125 to 31,825 of the sequence shown inSEQ ID NO:1 corresponding to an E3 deletion, and have nucleotides 35,643to 36,518 of the sequence shown in SEQ ID NO:1, and wherein thepartially deleted E4 gene is 5′ of the nucleotides 35,643 to 36,518.

The partially deleted E4 gene can be the E4 gene sequence shown in SEQID NO:1 that lacks at least nucleotides 34,916 to 35,642 of the sequenceshown in SEQ ID NO:1, nucleotides 2 to 34,916 of the sequence shown inSEQ ID NO:1, wherein the partially deleted E4 gene is 3′ of thenucleotides 2 to 34,916, the nucleotides 2 to 34,916 additionally lacknucleotides 577 to 3403 of the sequence shown in SEQ ID NO:1corresponding to an E1 deletion and lack nucleotides 27,125 to 31,825 ofthe sequence shown in SEQ ID NO:1 corresponding to an E3 deletion, andhave nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO:1,and wherein the partially deleted E4 gene is 5′ of the nucleotides35,643 to 36,518.

The adenovirus vector having the partially deleted E4 gene can have

Also disclosed herein is a method for delivering a cassette to amammalian cell comprising introducing into said cell an effective amountof a vector disclosed herein such as a C68 vector engineered toexpression the cassette.

Also disclosed herein is a method for producing a comprising introducinga vector disclosed herein into a mammalian cell, culturing the cellunder suitable conditions and producing the antigen.

V.E.2. E1-Expressing Complementation Cell Lines

To generate recombinant chimpanzee adenoviruses (Ad) deleted in any ofthe genes described herein, the function of the deleted gene region, ifessential to the replication and infectivity of the virus, can besupplied to the recombinant virus by a helper virus or cell line, i.e.,a complementation or packaging cell line. For example, to generate areplication-defective chimpanzee adenovirus vector, a cell line can beused which expresses the E1 gene products of the human or chimpanzeeadenovirus; such a cell line can include HEK293 or variants thereof. Theprotocol for the generation of the cell lines expressing the chimpanzeeE1 gene products (Examples 3 and 4 of U.S. Pat. No. 6,083,716) can befollowed to generate a cell line which expresses any selected chimpanzeeadenovirus gene.

An AAV augmentation assay can be used to identify a chimpanzeeadenovirus E1-expressing cell line. This assay is useful to identify E1function in cell lines made by using the E1 genes of otheruncharacterized adenoviruses, e.g., from other species. That assay isdescribed in Example 4B of U.S. Pat. No. 6,083,716.

A selected chimpanzee adenovirus gene, e.g., E1, can be under thetranscriptional control of a promoter for expression in a selectedparent cell line. Inducible or constitutive promoters can be employedfor this purpose. Among inducible promoters are included the sheepmetallothionine promoter, inducible by zinc, or the mouse mammary tumorvirus (MMTV) promoter, inducible by a glucocorticoid, particularly,dexamethasone. Other inducible promoters, such as those identified inInternational patent application WO95/13392, incorporated by referenceherein can also be used in the production of packaging cell lines.Constitutive promoters in control of the expression of the chimpanzeeadenovirus gene can be employed also.

A parent cell can be selected for the generation of a novel cell lineexpressing any desired C68 gene. Without limitation, such a parent cellline can be HeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No.CCL 185], KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38[CCL 75] cells. Other suitable parent cell lines can be obtained fromother sources. Parent cell lines can include CHO, HEK293 or variantsthereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a.

An E1-expressing cell line can be useful in the generation ofrecombinant chimpanzee adenovirus E1 deleted vectors. Cell linesconstructed using essentially the same procedures that express one ormore other chimpanzee adenoviral gene products are useful in thegeneration of recombinant chimpanzee adenovirus vectors deleted in thegenes that encode those products. Further, cell lines which expressother human Ad E1 gene products are also useful in generating chimpanzeerecombinant Ads.

V.E.3. Recombinant Viral Particles as Vectors

The compositions disclosed herein can comprise viral vectors, thatdeliver at least one antigen to cells. Such vectors comprise achimpanzee adenovirus DNA sequence such as C68 and a cassetteoperatively linked to regulatory sequences which direct expression ofthe cassette. The C68 vector is capable of expressing the cassette in aninfected mammalian cell. The C68 vector can be functionally deleted inone or more viral genes. A cassette comprises at least one antigen underthe control of one or more regulatory sequences such as a promoter.Optional helper viruses and/or packaging cell lines can supply to thechimpanzee viral vector any necessary products of deleted adenoviralgenes.

The term “functionally deleted” means that a sufficient amount of thegene region is removed or otherwise altered, e.g., by mutation ormodification, so that the gene region is no longer capable of producingone or more functional products of gene expression. Mutations ormodifications that can result in functional deletions include, but arenot limited to, nonsense mutations such as introduction of prematurestop codons and removal of canonical and non-canonical start codons,mutations that alter mRNA splicing or other transcriptional processing,or combinations thereof. If desired, the entire gene region can beremoved.

Modifications of the nucleic acid sequences forming the vectorsdisclosed herein, including sequence deletions, insertions, and othermutations may be generated using standard molecular biologicaltechniques and are within the scope of this invention.

V.E.4. Construction of the Viral Plasmid Vector

The chimpanzee adenovirus C68 vectors useful in this invention includerecombinant, defective adenoviruses, that is, chimpanzee adenovirussequences functionally deleted in the E1a or E1b genes, and optionallybearing other mutations, e.g., temperature-sensitive mutations ordeletions in other genes. It is anticipated that these chimpanzeesequences are also useful in forming hybrid vectors from otheradenovirus and/or adeno-associated virus sequences. Homologousadenovirus vectors prepared from human adenoviruses are described in thepublished literature [see, for example, Kozarsky I and II, cited above,and references cited therein, U.S. Pat. No. 5,240,846].

In the construction of useful chimpanzee adenovirus C68 vectors fordelivery of a cassette to a human (or other mammalian) cell, a range ofadenovirus nucleic acid sequences can be employed in the vectors. Avector comprising minimal chimpanzee C68 adenovirus sequences can beused in conjunction with a helper virus to produce an infectiousrecombinant virus particle. The helper virus provides essential geneproducts required for viral infectivity and propagation of the minimalchimpanzee adenoviral vector. When only one or more selected deletionsof chimpanzee adenovirus genes are made in an otherwise functional viralvector, the deleted gene products can be supplied in the viral vectorproduction process by propagating the virus in a selected packaging cellline that provides the deleted gene functions in trans.

V.E.5. Recombinant Minimal Adenovirus

A minimal chimpanzee Ad C68 virus is a viral particle containing justthe adenovirus cis-elements necessary for replication and virionencapsidation. That is, the vector contains the cis-acting 5′ and 3′inverted terminal repeat (ITR) sequences of the adenoviruses (whichfunction as origins of replication) and the native 5′ packaging/enhancerdomains (that contain sequences necessary for packaging linear Adgenomes and enhancer elements for the E1 promoter). See, for example,the techniques described for preparation of a “minimal” human Ad vectorin International Patent Application WO96/13597 and incorporated hereinby reference.

V.E.6. Other Defective Adenoviruses

Recombinant, replication-deficient adenoviruses can also contain morethan the minimal chimpanzee adenovirus sequences. These other Ad vectorscan be characterized by deletions of various portions of gene regions ofthe virus, and infectious virus particles formed by the optional use ofhelper viruses and/or packaging cell lines.

As one example, suitable vectors may be formed by deleting all or asufficient portion of the C68 adenoviral immediate early gene E1a anddelayed early gene E1b, so as to eliminate their normal biologicalfunctions. Replication-defective E1-deleted viruses are capable ofreplicating and producing infectious virus when grown on a chimpanzeeadenovirus-transformed, complementation cell line containing functionaladenovirus E1a and E1b genes which provide the corresponding geneproducts in trans. Based on the homologies to known adenovirussequences, it is anticipated that, as is true for the human recombinantE1-deleted adenoviruses of the art, the resulting recombinant chimpanzeeadenovirus is capable of infecting many cell types and can expressantigen(s), but cannot replicate in most cells that do not carry thechimpanzee E1 region DNA unless the cell is infected at a very highmultiplicity of infection.

As another example, all or a portion of the C68 adenovirus delayed earlygene E3 can be eliminated from the chimpanzee adenovirus sequence whichforms a part of the recombinant virus.

Chimpanzee adenovirus C68 vectors can also be constructed having adeletion of the E4 gene. Still another vector can contain a deletion inthe delayed early gene E2a.

Deletions can also be made in any of the late genes L1 through L5 of thechimpanzee C68 adenovirus genome. Similarly, deletions in theintermediate genes IX and IVa2 can be useful for some purposes. Otherdeletions may be made in the other structural or non-structuraladenovirus genes.

The above discussed deletions can be used individually, i.e., anadenovirus sequence can contain deletions of E1 only. Alternatively,deletions of entire genes or portions thereof effective to destroy orreduce their biological activity can be used in any combination. Forexample, in one exemplary vector, the adenovirus C68 sequence can havedeletions of the E1 genes and the E4 gene, or of the E1, E2a and E3genes, or of the E1 and E3 genes, or of E1, E2a and E4 genes, with orwithout deletion of E3, and so on. As discussed above, such deletionscan be used in combination with other mutations, such astemperature-sensitive mutations, to achieve a desired result.

The cassette comprising antigen(s) be inserted optionally into anydeleted region of the chimpanzee C68 Ad virus. Alternatively, thecassette can be inserted into an existing gene region to disrupt thefunction of that region, if desired.

V.E.7. Helper Viruses

Depending upon the chimpanzee adenovirus gene content of the viralvectors employed to carry the cassette, a helper adenovirus ornon-replicating virus fragment can be used to provide sufficientchimpanzee adenovirus gene sequences to produce an infective recombinantviral particle containing the cassette.

Useful helper viruses contain selected adenovirus gene sequences notpresent in the adenovirus vector construct and/or not expressed by thepackaging cell line in which the vector is transfected. A helper viruscan be replication-defective and contain a variety of adenovirus genesin addition to the sequences described above. The helper virus can beused in combination with the E1-expressing cell lines described herein.

For C68, the “helper” virus can be a fragment formed by clipping the Cterminal end of the C68 genome with SspI, which removes about 1300 bpfrom the left end of the virus. This clipped virus is thenco-transfected into an E1-expressing cell line with the plasmid DNA,thereby forming the recombinant virus by homologous recombination withthe C68 sequences in the plasmid.

Helper viruses can also be formed into poly-cation conjugates asdescribed in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helpervirus can optionally contain a reporter gene. A number of such reportergenes are known to the art. The presence of a reporter gene on thehelper virus which is different from the cassette on the adenovirusvector allows both the Ad vector and the helper virus to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

V.E.8. Assembly of Viral Particle and Infection of a Cell Line

Assembly of the selected DNA sequences of the adenovirus, the cassette,and other vector elements into various intermediate plasmids and shuttlevectors, and the use of the plasmids and vectors to produce arecombinant viral particle can all be achieved using conventionaltechniques. Such techniques include conventional cloning techniques ofcDNA, in vitro recombination techniques (e.g., Gibson assembly), use ofoverlapping oligonucleotide sequences of the adenovirus genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleic acid sequence. Standard transfection and co-transfectiontechniques are employed, e.g., CaPO4 precipitation techniques orliposome-mediated transfection methods such as lipofectamine. Otherconventional methods employed include homologous recombination of theviral genomes, plaquing of viruses in agar overlay, methods of measuringsignal generation, and the like.

For example, following the construction and assembly of the desiredcassette-containing viral vector, the vector can be transfected in vitroin the presence of a helper virus into the packaging cell line.Homologous recombination occurs between the helper and the vectorsequences, which permits the adenovirus-antigen sequences in the vectorto be replicated and packaged into virion capsids, resulting in therecombinant viral vector particles.

The resulting recombinant chimpanzee C68 adenoviruses are useful intransferring a cassette to a selected cell. In in vivo experiments withthe recombinant virus grown in the packaging cell lines, the E1-deletedrecombinant chimpanzee adenovirus demonstrates utility in transferring acassette to a non-chimpanzee, preferably a human, cell.

V.E.9. Use of the Recombinant Virus Vectors

The resulting recombinant chimpanzee C68 adenovirus containing thecassette (produced by cooperation of the adenovirus vector and helpervirus or adenoviral vector and packaging cell line, as described above)thus provides an efficient gene transfer vehicle which can deliverantigen(s) to a subject in vivo or ex vivo.

The above-described recombinant vectors are administered to humansaccording to published methods for gene therapy. A chimpanzee viralvector bearing a cassette can be administered to a patient, preferablysuspended in a biologically compatible solution or pharmaceuticallyacceptable delivery vehicle. A suitable vehicle includes sterile saline.Other aqueous and non-aqueous isotonic sterile injection solutions andaqueous and non-aqueous sterile suspensions known to be pharmaceuticallyacceptable carriers and well known to those of skill in the art may beemployed for this purpose.

The chimpanzee adenoviral vectors are administered in sufficient amountsto transduce the human cells and to provide sufficient levels of antigentransfer and expression to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the liver, intranasal, intravenous,intramuscular, subcutaneous, intradermal, oral and other parental routesof administration. Routes of administration may be combined, if desired.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. The dosage will be adjusted to balance thetherapeutic benefit against any side effects and such dosages may varydepending upon the therapeutic application for which the recombinantvector is employed. The levels of expression of antigen(s) can bemonitored to determine the frequency of dosage administration.

Recombinant, replication defective adenoviruses can be administered in a“pharmaceutically effective amount”, that is, an amount of recombinantadenovirus that is effective in a route of administration to transfectthe desired cells and provide sufficient levels of expression of theselected gene to provide a vaccinal benefit, i.e., some measurable levelof protective immunity. C68 vectors comprising a cassette can beco-administered with adjuvant. Adjuvant can be separate from the vector(e.g., alum) or encoded within the vector, in particular if the adjuvantis a protein. Adjuvants are well known in the art.

Conventional and pharmaceutically acceptable routes of administrationinclude, but are not limited to, intranasal, intramuscular,intratracheal, subcutaneous, intradermal, rectal, oral and otherparental routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the immunogen or thedisease. For example, in prophylaxis of rabies, the subcutaneous,intratracheal and intranasal routes are preferred. The route ofadministration primarily will depend on the nature of the disease beingtreated.

The levels of immunity to antigen(s) can be monitored to determine theneed, if any, for boosters. Following an assessment of antibody titersin the serum, for example, optional booster immunizations may be desired

VI. Therapeutic and Manufacturing Methods

Also provided is a method of inducing a tumor specific immune responsein a subject, vaccinating against a tumor, treating and/or alleviating asymptom of cancer in a subject by administering to the subject one ormore antigens such as a plurality of antigens identified using methodsdisclosed herein.

Also provided is a method of inducing an infectious diseaseorganism-specific immune response in a subject, vaccinating against aninfectious disease organism, treating and/or alleviating a symptom of aninfection associated with an infectious disease organism in a subject byadministering to the subject one or more antigens such as a plurality ofantigens identified using methods disclosed herein.

In some aspects, a subject has been diagnosed with cancer or is at riskof developing cancer. A subject can be a human, dog, cat, horse or anyanimal in which a tumor specific immune response is desired. A tumor canbe any solid tumor such as breast, ovarian, prostate, lung, kidney,gastric, colon, testicular, head and neck, pancreas, brain, melanoma,and other tumors of tissue organs and hematological tumors, such aslymphomas and leukemias, including acute myelogenous leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocyticleukemia, and B cell lymphomas.

In some aspects, a subject has been diagnosed with an infection or is atrisk of an infection (e.g., age, geographical/travel, and/orwork-related increased risk of or predisposition to an infection, or atrisk to a seasonal and/or novel disease infection).

An antigen can be administered in an amount sufficient to induce a CTLresponse. An antigen can be administered in an amount sufficient toinduce a T cell response. An antigen can be administered in an amountsufficient to induce a B cell response.

An antigen can be administered alone or in combination with othertherapeutic agents. The therapeutic agent is for example, achemotherapeutic agent, radiation, or immunotherapy. Any suitabletherapeutic treatment for a particular cancer can be administered.Therapeutic agents can include those that target an infectious diseaseorganism, such as an anti-viral or antibiotic agent.

In addition, a subject can be further administered ananti-immunosuppressive/immunostimulatory agent such as a checkpointinhibitor. For example, the subject can be further administered ananti-CTLA antibody or anti-PD-1 or anti-PD-L1. Blockade of CTLA-4 orPD-L1 by antibodies can enhance the immune response to cancerous cellsin the patient. In particular, CTLA-4 blockade has been shown effectivewhen following a vaccination protocol.

The optimum amount of each antigen to be included in a vaccinecomposition and the optimum dosing regimen can be determined. Forexample, an antigen or its variant can be prepared for intravenous(i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.)injection, intraperitoneal (i.p.) injection, intramuscular (i.m.)injection. Methods of injection include s.c., i.d., i.p., i.m., and i.v.Methods of DNA or RNA injection include i.d., i.m., s.c., i.p. and i.v.Other methods of administration of the vaccine composition are known tothose skilled in the art.

A vaccine can be compiled so that the selection, number and/or amount ofantigens present in the composition is/are tissue, cancer, infectiousdisease, and/or patient-specific. For instance, the exact selection ofpeptides can be guided by expression patterns of the parent proteins ina given tissue or guided by mutation or disease status of a patient. Theselection can be dependent on the specific type of cancer, the specificinfectious disease, the status of the disease, the goal of thevaccination (e.g., preventative or targeting an ongoing disease),earlier treatment regimens, the immune status of the patient, and, ofcourse, the HLA-haplotype of the patient. Furthermore, a vaccine cancontain individualized components, according to personal needs of theparticular patient. Examples include varying the selection of antigensaccording to the expression of the antigen in the particular patient oradjustments for secondary treatments following a first round or schemeof treatment.

A patient can be identified for administration of an antigen vaccinethrough the use of various diagnostic methods, e.g., patient selectionmethods described further below. Patient selection can involveidentifying mutations in, or expression patterns of, one or more genes.Patient selection can involve identifying the infectious disease of anongoing infection. Patient selection can involve identifying risk of aninfection by an infectious disease. In some cases, patient selectioninvolves identifying the haplotype of the patient. The various patientselection methods can be performed in parallel, e.g., a sequencingdiagnostic can identify both the mutations and the haplotype of apatient. The various patient selection methods can be performedsequentially, e.g., one diagnostic test identifies the mutations andseparate diagnostic test identifies the haplotype of a patient, andwhere each test can be the same (e.g., both high-throughput sequencing)or different (e.g., one high-throughput sequencing and the other Sangersequencing) diagnostic methods.

For a composition to be used as a vaccine for cancer or an infectiousdisease, antigens with similar normal self-peptides that are expressedin high amounts in normal tissues can be avoided or be present in lowamounts in a composition described herein. On the other hand, if it isknown that the tumor or infected cell of a patient expresses highamounts of a certain antigen, the respective pharmaceutical compositionfor treatment of this cancer or infection can be present in high amountsand/or more than one antigen specific for this particularly antigen orpathway of this antigen can be included.

Compositions comprising an antigen can be administered to an individualalready suffering from cancer or an infection. In therapeuticapplications, compositions are administered to a patient in an amountsufficient to elicit an effective CTL response to the tumor antigen orinfectious disease organism antigen and to cure or at least partiallyarrest symptoms and/or complications. An amount adequate to accomplishthis is defined as “therapeutically effective dose.” Amounts effectivefor this use will depend on, e.g., the composition, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician. It should be kept in mind that compositionscan generally be employed in serious disease states, that is,life-threatening or potentially life threatening situations, especiallywhen the cancer has metastasized. In such cases, in view of theminimization of extraneous substances and the relative nontoxic natureof an antigen, it is possible and can be felt desirable by the treatingphysician to administer substantial excesses of these compositions.

For therapeutic use, administration can begin at the detection orsurgical removal of tumors or begin at the detection or treatment of aninfection. This can be followed by boosting doses until at leastsymptoms are substantially abated and for a period thereafter.

The pharmaceutical compositions (e.g., vaccine compositions) fortherapeutic treatment are intended for parenteral, topical, nasal, oralor local administration. A pharmaceutical compositions can beadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. The compositions can be administeredat the site of surgical excision to induce a local immune response tothe tumor. The compositions can be administered to target specificinfected tissues and/or cells (e.g., antigen presenting cells) of asubject. Disclosed herein are compositions for parenteral administrationwhich comprise a solution of the antigen and vaccine compositions aredissolved or suspended in an acceptable carrier, e.g., an aqueouscarrier. A variety of aqueous carriers can be used, e.g., water,buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.These compositions can be sterilized by conventional, well knownsterilization techniques, or can be sterile filtered. The resultingaqueous solutions can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

Antigens can also be administered via liposomes, which target them to aparticular cells tissue, such as lymphoid tissue. Liposomes are alsouseful in increasing half-life. Liposomes include emulsions, foams,micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations theantigen to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to, e.g., a receptorprevalent among lymphoid cells, such as monoclonal antibodies which bindto the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes filled with a desired antigen can bedirected to the site of lymphoid cells, where the liposomes then deliverthe selected therapeutic/immunogenic compositions. Liposomes can beformed from standard vesicle-forming lipids, which generally includeneutral and negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728,4,501,728, 4,837,028, and 5,019,369.

For targeting to the immune cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the desired immune system cells. A liposomesuspension can be administered intravenously, locally, topically, etc.in a dose which varies according to, inter alia, the manner ofadministration, the peptide being delivered, and the stage of thedisease being treated.

For therapeutic or immunization purposes, nucleic acids encoding apeptide and optionally one or more of the peptides described herein canalso be administered to the patient. A number of methods areconveniently used to deliver the nucleic acids to the patient. Forinstance, the nucleic acid can be delivered directly, as “naked DNA”.This approach is described, for instance, in Wolff et al., Science 247:1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466. Thenucleic acids can also be administered using ballistic delivery asdescribed, for instance, in U.S. Pat. No. 5,204,253. Particles comprisedsolely of DNA can be administered. Alternatively, DNA can be adhered toparticles, such as gold particles. Approaches for delivering nucleicacid sequences can include viral vectors, mRNA vectors, and DNA vectorswith or without electroporation.

The nucleic acids can also be delivered complexed to cationic compounds,such as cationic lipids. Lipid-mediated gene delivery methods aredescribed, for instance, in 9618372WOAWO 96/18372; 9324640WOAWO93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988);U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; 9106309WOAWO91/06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414(1987).

Antigens can also be included in viral vector-based vaccine platforms,such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus,adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy(2004) 10, 616-629), or lentivirus, including but not limited to second,third or hybrid second/third generation lentivirus and recombinantlentivirus of any generation designed to target specific cell types orreceptors (See, e.g., Hu et al., Immunization Delivered by LentiviralVectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61, Sakuma et al., Lentiviral vectors: basic to translational,Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue ofsplicing-mediated intron loss maximizes expression in lentiviral vectorscontaining the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector forSafe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12):9873-9880). Dependent on the packaging capacity of the above mentionedviral vector-based vaccine platforms, this approach can deliver one ormore nucleic acid sequences that encode one or more antigen peptides.The sequences may be flanked by non-mutated sequences, may be separatedby linkers or may be preceded with one or more sequences targeting asubcellular compartment (See, e.g., Gros et al., Prospectiveidentification of neoantigen-specific lymphocytes in the peripheralblood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen etal., Targeting of cancer neoantigens with donor-derived T cell receptorrepertoires, Science. (2016) 352 (6291):1337-41, Lu et al., Efficientidentification of mutated cancer antigens recognized by T cellsassociated with durable tumor regressions, Clin Cancer Res. (2014)20(13):3401-10). Upon introduction into a host, infected cells expressthe antigens, and thereby elicit a host immune (e.g., CTL) responseagainst the peptide(s). Vaccinia vectors and methods useful inimmunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.Another vector is BCG (Bacille Calmette Guerin). BCG vectors aredescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vaccine vectors useful for therapeutic administration orimmunization of antigens, e.g., Salmonella typhi vectors, and the likewill be apparent to those skilled in the art from the descriptionherein.

A means of administering nucleic acids uses minigene constructs encodingone or multiple epitopes. To create a DNA sequence encoding the selectedCTL epitopes (minigene) for expression in human cells, the amino acidsequences of the epitopes are reverse translated. A human codon usagetable is used to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences are directly adjoined, creating acontinuous polypeptide sequence. To optimize expression and/orimmunogenicity, additional elements can be incorporated into theminigene design. Examples of amino acid sequence that could be reversetranslated and included in the minigene sequence include: helper Tlymphocyte, epitopes, a leader (signal) sequence, and an endoplasmicreticulum retention signal. In addition, MHC presentation of CTLepitopes can be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL epitopes. Theminigene sequence is converted to DNA by assembling oligonucleotidesthat encode the plus and minus strands of the minigene. Overlappingoligonucleotides (30-100 bases long) are synthesized, phosphorylated,purified and annealed under appropriate conditions using well knowntechniques. The ends of the oligonucleotides are joined using T4 DNAligase. This synthetic minigene, encoding the CTL epitope polypeptide,can then cloned into a desired expression vector.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). A variety of methods have beendescribed, and new techniques can become available. As noted above,nucleic acids are conveniently formulated with cationic lipids. Inaddition, glycolipids, fusogenic liposomes, peptides and compoundsreferred to collectively as protective, interactive, non-condensing(PINC) could also be complexed to purified plasmid DNA to influencevariables such as stability, intramuscular dispersion, or trafficking tospecific organs or cell types.

Also disclosed is a method of manufacturing a vaccine, comprisingperforming the steps of a method disclosed herein; and producing avaccine comprising a plurality of antigens or a subset of the pluralityof antigens. Also disclosed is a method of manufacturing adenoviralvector, comprising performing the steps of a method disclosed herein;and producing an adenoviral vector comprising a cassette. For example,disclosed is a method of manufacturing adenoviral vector using a TETpromoter system, such as the TETr controlled promoter system describedherein. Viral production using the TETr controlled promoter system caninclude a. providing a viral vector comprising a cassette, the cassettecomprising: (i) at least one payload nucleic acid sequence, and (ii) atleast one promoter sequence operably linked to the at least one payloadnucleic acid sequence, wherein the at least one promoter is atetracycline (TET) repressor protein (TETr) controlled promoter, b.providing a cell engineered to express the TETr protein; and c.contacting the viral vector with the cell under conditions sufficientfor production of the virus.

Antigens disclosed herein can be manufactured using methods known in theart. For example, a method of producing an antigen or a vector (e.g., avector including at least one sequence encoding one or more antigens)disclosed herein can include culturing a host cell under conditionssuitable for expressing the antigen or vector wherein the host cellcomprises at least one polynucleotide encoding the antigen or vector,and purifying the antigen or vector. Standard purification methodsinclude chromatographic techniques, electrophoretic, immunological,precipitation, dialysis, filtration, concentration, and chromatofocusingtechniques.

Host cells can include a Chinese Hamster Ovary (CHO) cell, NS0 cell,yeast, or a HEK293 cell. Host cells can be transformed with one or morepolynucleotides comprising at least one nucleic acid sequence thatencodes an antigen or vector disclosed herein, optionally wherein theisolated polynucleotide further comprises a promoter sequence operablylinked to the at least one nucleic acid sequence that encodes theantigen or vector. In certain embodiments the isolated polynucleotidecan be cDNA.

VII. Antigen Use and Administration

A vaccination protocol can be used to dose a subject with one or moreantigens. A priming vaccine and a boosting vaccine can be used to dosethe subject. The priming vaccine can be based on C68 (e.g., thesequences shown in SEQ ID NO:1 or 2) or srRNA (e.g., the sequences shownin SEQ ID NO:3 or 4) and the boosting vaccine can be based on C68 (e.g.,the sequences shown in SEQ ID NO:1 or 2) or srRNA (e.g., the sequencesshown in SEQ ID NO:3 or 4). Each vector typically includes a cassettethat includes antigens. Cassettes can include about 20 antigens,separated by spacers such as the natural sequence that normallysurrounds each antigen or other non-natural spacer sequences such asAAY. Cassettes can also include MHCII antigens such a tetanus toxoidantigen and PADRE antigen, which can be considered universal class IIantigens. Cassettes can also include a targeting sequence such as aubiquitin targeting sequence. In addition, each vaccine dose can beadministered to the subject in conjunction with (e.g., concurrently,before, or after) a checkpoint inhibitor (CPI). CPI's can include thosethat inhibit CTLA4, PD1, and/or PDL1 such as antibodies orantigen-binding portions thereof. Such antibodies can includetremelimumab or durvalumab.

A priming vaccine can be injected (e.g., intramuscularly) in a subject.Bilateral injections per dose can be used. For example, one or moreinjections of ChAdV68 (C68) can be used (e.g., total dose 1×10¹² viralparticles); one or more injections of self-replicating RNA (srRNA) atlow vaccine dose selected from the range 0.001 to 1 ug RNA, inparticular 0.1 or 1 ug can be used; or one or more injections of srRNAat high vaccine dose selected from the range 1 to 100 ug RNA, inparticular 10 or 100 ug can be used.

A vaccine boost (boosting vaccine) can be injected (e.g.,intramuscularly) after prime vaccination. A boosting vaccine can beadministered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, e.g.,every 4 weeks and/or 8 weeks after the prime. Bilateral injections perdose can be used. For example, one or more injections of ChAdV68 (C68)can be used (e.g., total dose 1×10¹² viral particles); one or moreinjections of self-replicating RNA (srRNA) at low vaccine dose selectedfrom the range 0.001 to 1 ug RNA, in particular 0.1 or 1 ug can be used;or one or more injections of srRNA at high vaccine dose selected fromthe range 1 to 100 ug RNA, in particular 10 or 100 ug can be used.

Anti-CTLA-4 (e.g., tremelimumab) can also be administered to thesubject. For example, anti-CTLA4 can be administered subcutaneously nearthe site of the intramuscular vaccine injection (ChAdV68 prime or srRNAlow doses) to ensure drainage into the same lymph node. Tremelimumab isa selective human IgG2 mAb inhibitor of CTLA-4. Target Anti-CTLA-4(tremelimumab) subcutaneous dose is typically 70-75 mg (in particular 75mg) with a dose range of, e.g., 1-100 mg or 5-420 mg.

In certain instances an anti-PD-L1 antibody can be used such asdurvalumab (MEDI 4736). Durvalumab is a selective, high affinity humanIgGI mAb that blocks PD-L1 binding to PD-1 and CD80. Durvalumab isgenerally administered at 20 mg/kg i.v. every 4 weeks.

Immune monitoring can be performed before, during, and/or after vaccineadministration. Such monitoring can inform safety and efficacy, amongother parameters.

To perform immune monitoring, PBMCs are commonly used. PBMCs can beisolated before prime vaccination, and after prime vaccination (e.g. 4weeks and 8 weeks). PBMCs can be harvested just prior to boostvaccinations and after each boost vaccination (e.g. 4 weeks and 8weeks).

T cell responses can be assessed as part of an immune monitoringprotocol. For example, the ability of a vaccine composition describedherein to stimulate an immune response can be monitored and/or assessed.As used herein, “stimulate an immune response” refers to any increase inan immune response, such as initiating an immune response (e.g., apriming vaccine stimulating the initiation of an immune response in anaïve subject) or enhancement of an immune response (e.g., a boostingvaccine stimulating the enhancement of an immune response in a subjecthaving a pre-existing immune response to an antigen, such as apre-existing immune response initiated by a priming vaccine). T cellresponses can be measured using one or more methods known in the artsuch as ELISpot, intracellular cytokine staining, cytokine secretion andcell surface capture, T cell proliferation, MHC multimer staining, or bycytotoxicity assay. T cell responses to epitopes encoded in vaccines canbe monitored from PBMCs by measuring induction of cytokines, such asIFN-gamma, using an ELISpot assay. Specific CD4 or CD8 T cell responsesto epitopes encoded in vaccines can be monitored from PBMCs by measuringinduction of cytokines captured intracellularly or extracellularly, suchas IFN-gamma, using flow cytometry. Specific CD4 or CD8 T cell responsesto epitopes encoded in the vaccines can be monitored from PBMCs bymeasuring T cell populations expressing T cell receptors specific forepitope/MHC class I complexes using MHC multimer staining. Specific CD4or CD8 T cell responses to epitopes encoded in the vaccines can bemonitored from PBMCs by measuring the ex vivo expansion of T cellpopulations following 3H-thymidine, bromodeoxyuridine andcarboxyfluoresceine-diacetate-succinimidylester (CFSE) incorporation.The antigen recognition capacity and lytic activity of PBMC-derived Tcells that are specific for epitopes encoded in vaccines can be assessedfunctionally by chromium release assay or alternative colorimetriccytotoxicity assays.

B cell responses can be measured using one or more methods known in theart such as assays used to determine B cell differentiation (e.g.,differentiation into plasma cells), B cell or plasma cell proliferation,B cell or plasma cell activation (e.g., upregulation of costimulatorymarkers such as CD80 or CD86), antibody class switching, and/or antibodyproduction (e.g., an ELISA).

VIII. Antigen Identification

VIII.A. Antigen Candidate Identification

Research methods for NGS analysis of tumor and normal exome andtranscriptomes have been described and applied in the antigenidentification space.^(6,14,15) Certain optimizations for greatersensitivity and specificity for antigen identification in the clinicalsetting can be considered. These optimizations can be grouped into twoareas, those related to laboratory processes and those related to theNGS data analysis. The research methods described can also be applied toidentification of antigens in other settings, such as identification ofidentifying antigens from an infectious disease organism, an infectionin a subject, or an infected cell of a subject. Examples ofoptimizations are known to those skilled in the art, for example themethods described in more detail in U.S. Pat. No. 10,055,540, USApplication Pub. No. US20200010849A1, and international patentapplication publications WO/2018/195357 and WO/2018/208856, each hereinincorporated by reference, in their entirety, for all purposes.

VIII.B. Isolation and Detection of HLA Peptides

Isolation of HLA-peptide molecules was performed using classicimmunoprecipitation (IP) methods after lysis and solubilization of thetissue sample (55-58). A clarified lysate was used for HLA specific IP.

Immunoprecipitation was performed using antibodies coupled to beadswhere the antibody is specific for HLA molecules. For a pan-Class I HLAimmunoprecipitation, a pan-Class I CR antibody is used, for Class IIHLA-DR, an HLA-DR antibody is used. Antibody is covalently attached toNHS-sepharose beads during overnight incubation. After covalentattachment, the beads were washed and aliquoted for IP. (59, 60)Immunoprecipitations can also be performed with antibodies that are notcovalently attached to beads. Typically this is done using sepharose ormagnetic beads coated with Protein A and/or Protein G to hold theantibody to the column. Some antibodies that can be used to selectivelyenrich MHC/peptide complex are listed below.

Antibody Name Specificity W6/32 Class I HLA-A, B, C L243 Class II-HLA-DRTu36 Class II-HLA-DR LN3 Class II-HLA-DR Tu39 Class II-HLA-DR, DP, DQ

The clarified tissue lysate is added to the antibody beads for theimmunoprecipitation. After immunoprecipitation, the beads are removedfrom the lysate and the lysate stored for additional experiments,including additional IPs. The IP beads are washed to remove non-specificbinding and the HLA/peptide complex is eluted from the beads usingstandard techniques. The protein components are removed from thepeptides using a molecular weight spin column or C18 fractionation. Theresultant peptides are taken to dryness by SpeedVac evaporation and insome instances are stored at −20 C prior to MS analysis.

Dried peptides are reconstituted in an HPLC buffer suitable for reversephase chromatography and loaded onto a C-18 microcapillary HPLC columnfor gradient elution in a Fusion Lumos mass spectrometer (Thermo). MS1spectra of peptide mass/charge (m/z) were collected in the Orbitrapdetector at high resolution followed by MS2 low resolution scanscollected in the ion trap detector after HCD fragmentation of theselected ion. Additionally, MS2 spectra can be obtained using either CIDor ETD fragmentation methods or any combination of the three techniquesto attain greater amino acid coverage of the peptide. MS2 spectra canalso be measured with high resolution mass accuracy in the Orbitrapdetector.

MS2 spectra from each analysis are searched against a protein databaseusing Comet (61, 62) and the peptide identification are scored usingPercolator (63-65). Additional sequencing is performed using PEAKSstudio (Bioinformatics Solutions Inc.) and other search engines orsequencing methods can be used including spectral matching and de novosequencing (97).

VIII.B.1. MS Limit of Detection Studies in Support of Comprehensive HLAPeptide Sequencing.

Using the peptide YVYVADVAAK (SEQ ID NO: 77) it was determined what thelimits of detection are using different amounts of peptide loaded ontothe LC column. The amounts of peptide tested were 1 pmol, 100 fmol, 10fmol, 1 fmol, and 100 amol. (Table 1) The results are shown in FIGS. 24Aand 24B. These results indicate that the lowest limit of detection (LoD)is in the attomol range (10⁻¹⁸), that the dynamic range spans fiveorders of magnitude, and that the signal to noise appears sufficient forsequencing at low femtomol ranges (10⁻¹⁵).

TABLE 1 Peptide Loaded Copies/Cell m/z on Column in 1e9cells 566.830 1pmol 600 562.823 100 fmol 60 559.816 10 fmol 6 556.810 1 fmol 0.6553.802 100 amol 0.06

IX. Presentation Model

Presentation models can be used to identify likelihoods of peptidepresentation in patients. Various presentation models are known to thoseskilled in the art, for example the presentation models described inmore detail in U.S. Pat. No. 10,055,540, US Application Pub. No.US20200010849A1 and US20110293637, and international patent applicationpublications WO/2018/195357, WO/2018/208856, and WO2016187508, eachherein incorporated by reference, in their entirety, for all purposes.

X. Training Module

Training modules can be used to construct one or more presentationmodels based on training data sets that generate likelihoods of whetherpeptide sequences will be presented by MHC alleles associated with thepeptide sequences. Various training modules are known to those skilledin the art, for example the presentation models described in more detailin U.S. Pat. No. 10,055,540, US Application Pub. No. US20200010849A1,and international patent application publications WO/2018/195357, andWO/2018/208856, each herein incorporated by reference, in theirentirety, for all purposes. A training module can construct apresentation model to predict presentation likelihoods of peptides on aper-allele basis. A training module can also construct a presentationmodel to predict presentation likelihoods of peptides in amultiple-allele setting where two or more MHC alleles are present.

XI. Prediction Module

A prediction module can be used to receive sequence data and selectcandidate antigens in the sequence data using a presentation model.Specifically, the sequence data may be DNA sequences, RNA sequences,and/or protein sequences extracted from tumor tissue cells of patients,infected cells patients, or infectious disease organisms themselves. Aprediction module may identify candidate neoantigens that are mutatedpeptide sequences by comparing sequence data extracted from normaltissue cells of a patient with the sequence data extracted from tumortissue cells of the patient to identify portions containing one or moremutations. A prediction module may identify candidate antigens that arepathogen-derived peptides, virally-derived peptides, bacterially-derivedpeptides, fungally-derived peptides, and parasitically-derived peptides,such as by comparing sequence data extracted from normal tissue cells ofa patient with the sequence data extracted from infected cells of thepatient to identify portions containing one or more infectious diseaseorganism associated antigens. A prediction module may identify candidateantigens that have altered expression in a tumor cell or canceroustissue in comparison to a normal cell or tissue by comparing sequencedata extracted from normal tissue cells of a patient with the sequencedata extracted from tumor tissue cells of the patient to identifyimproperly expressed candidate antigens. A prediction module mayidentify candidate antigens that are expressed in an infected cell orinfected tissue in comparison to a normal cell or tissue by comparingsequence data extracted from normal tissue cells of a patient with thesequence data extracted from infected tissue cells of the patient toidentify expressed candidate antigens (e.g., identifying expressedpolynucleotides and/or polypeptides specific to an infectious disease).

A presentation module can apply one or more presentation model toprocessed peptide sequences to estimate presentation likelihoods of thepeptide sequences. Specifically, the prediction module may select one ormore candidate antigen peptide sequences that are likely to be presentedon tumor HLA molecules or infected cell HLA molecules by applyingpresentation models to the candidate antigens. In one implementation,the presentation module selects candidate antigen sequences that haveestimated presentation likelihoods above a predetermined threshold. Inanother implementation, the presentation model selects the N candidateantigen sequences that have the highest estimated presentationlikelihoods (where N is generally the maximum number of epitopes thatcan be delivered in a vaccine). A vaccine including the selectedcandidate antigens for a given patient can be injected into the patientto induce immune responses.

XI.B. Cassette Design Module

XI.B.1 Overview

A cassette design module can be used to generate a vaccine cassettesequence based on selected candidate peptides for injection into apatient. Various cassette design modules are known to those skilled inthe art, for example the cassette design modules described in moredetail in U.S. Pat. No. 10,055,540, US Application Pub. No.US20200010849A1, and international patent application publicationsWO/2018/195357 and WO/2018/208856, each herein incorporated byreference, in their entirety, for all purposes.

A set of therapeutic epitopes may be generated based on the selectedpeptides determined by a prediction module associated with presentationlikelihoods above a predetermined threshold, where the presentationlikelihoods are determined by the presentation models. However it isappreciated that in other embodiments, the set of therapeutic epitopesmay be generated based on any one or more of a number of methods (aloneor in combination), for example, based on binding affinity or predictedbinding affinity to HLA class I or class II alleles of the patient,binding stability or predicted binding stability to HLA class I or classII alleles of the patient, random sampling, and the like.

Therapeutic epitopes may correspond to selected peptides themselves.Therapeutic epitopes may also include C- and/or N-terminal flankingsequences in addition to the selected peptides. N- and C-terminalflanking sequences can be the native N- and C-terminal flankingsequences of the therapeutic vaccine epitope in the context of itssource protein. Therapeutic epitopes can represent a fixed-lengthepitope Therapeutic epitopes can represent a variable-length epitope, inwhich the length of the epitope can be varied depending on, for example,the length of the C- or N-flanking sequence. For example, the C-terminalflanking sequence and the N-terminal flanking sequence can each havevarying lengths of 2-5 residues, resulting in 16 possible choices forthe epitope.

A cassette design module can also generate cassette sequences by takinginto account presentation of junction epitopes that span the junctionbetween a pair of therapeutic epitopes in the cassette. Junctionepitopes are novel non-self but irrelevant epitope sequences that arisein the cassette due to the process of concatenating therapeutic epitopesand linker sequences in the cassette. The novel sequences of junctionepitopes are different from the therapeutic epitopes of the cassettethemselves.

A cassette design module can generate a cassette sequence that reducesthe likelihood that junction epitopes are presented in the patient.Specifically, when the cassette is injected into the patient, junctionepitopes have the potential to be presented by HLA class I or HLA classII alleles of the patient, and stimulate a CD8 or CD4 T-cell response,respectively. Such reactions are often times undesirable because T-cellsreactive to the junction epitopes have no therapeutic benefit, and maydiminish the immune response to the selected therapeutic epitopes in thecassette by antigenic competition.⁷⁶

A cassette design module can iterate through one or more candidatecassettes, and determine a cassette sequence for which a presentationscore of junction epitopes associated with that cassette sequence isbelow a numerical threshold. The junction epitope presentation score isa quantity associated with presentation likelihoods of the junctionepitopes in the cassette, and a higher value of the junction epitopepresentation score indicates a higher likelihood that junction epitopesof the cassette will be presented by HLA class I or HLA class II orboth.

In one embodiment, a cassette design module may determine a cassettesequence associated with the lowest junction epitope presentation scoreamong the candidate cassette sequences.

A cassette design module may iterate through one or more candidatecassette sequences, determine the junction epitope presentation scorefor the candidate cassettes, and identify an optimal cassette sequenceassociated with a junction epitope presentation score below thethreshold.

A cassette design module may further check the one or more candidatecassette sequences to identify if any of the junction epitopes in thecandidate cassette sequences are self-epitopes for a given patient forwhom the vaccine is being designed. To accomplish this, the cassettedesign module checks the junction epitopes against a known database suchas BLAST. In one embodiment, the cassette design module may beconfigured to design cassettes that avoid junction self-epitopes.

A cassette design module can perform a brute force approach and iteratethrough all or most possible candidate cassette sequences to select thesequence with the smallest junction epitope presentation score. However,the number of such candidate cassettes can be prohibitively large as thecapacity of the vaccine increases. For example, for a vaccine capacityof 20 epitopes, the cassette design module has to iterate through ˜10¹⁸possible candidate cassettes to determine the cassette with the lowestjunction epitope presentation score. This determination may becomputationally burdensome (in terms of computational processingresources required), and sometimes intractable, for the cassette designmodule to complete within a reasonable amount of time to generate thevaccine for the patient. Moreover, accounting for the possible junctionepitopes for each candidate cassette can be even more burdensome. Thus,a cassette design module may select a cassette sequence based on ways ofiterating through a number of candidate cassette sequences that aresignificantly smaller than the number of candidate cassette sequencesfor the brute force approach.

A cassette design module can generate a subset of randomly or at leastpseudo-randomly generated candidate cassettes, and selects the candidatecassette associated with a junction epitope presentation score below apredetermined threshold as the cassette sequence. Additionally, thecassette design module may select the candidate cassette from the subsetwith the lowest junction epitope presentation score as the cassettesequence. For example, the cassette design module may generate a subsetof ˜1 million candidate cassettes for a set of 20 selected epitopes, andselect the candidate cassette with the smallest junction epitopepresentation score. Although generating a subset of random cassettesequences and selecting a cassette sequence with a low junction epitopepresentation score out of the subset may be sub-optimal relative to thebrute force approach, it requires significantly less computationalresources thereby making its implementation technically feasible.Further, performing the brute force method as opposed to this moreefficient technique may only result in a minor or even negligibleimprovement injunction epitope presentation score, thus making it notworthwhile from a resource allocation perspective. A cassette designmodule can determine an improved cassette configuration by formulatingthe epitope sequence for the cassette as an asymmetric travelingsalesman problem (TSP). Given a list of nodes and distances between eachpair of nodes, the TSP determines a sequence of nodes associated withthe shortest total distance to visit each node exactly once and returnto the original node. For example, given cities A, B, and C with knowndistances between each other, the solution of the TSP generates a closedsequence of cities, for which the total distance traveled to visit eachcity exactly once is the smallest among possible routes. The asymmetricversion of the TSP determines the optimal sequence of nodes when thedistance between a pair of nodes are asymmetric. For example, the“distance” for traveling from node A to node B may be different from the“distance” for traveling from node B to node A. By solving for animproved optimal cassette using an asymmetric TSP, the cassette designmodule can find a cassette sequence that results in a reducedpresentation score across the junctions between epitopes of thecassette. The solution of the asymmetric TSP indicates a sequence oftherapeutic epitopes that correspond to the order in which the epitopesshould be concatenated in a cassette to minimize the junction epitopepresentation score across the junctions of the cassette. A cassettesequence determined through this approach can result in a sequence withsignificantly less presentation of junction epitopes while potentiallyrequiring significantly less computational resources than the randomsampling approach, especially when the number of generated candidatecassette sequences is large. Illustrative examples of differentcomputational approaches and comparisons for optimizing cassette designare described in more detail in U.S. Pat. No. 10,055,540, US ApplicationPub. No. US20200010849A1, and international patent applicationpublications WO/2018/195357 and WO/2018/208856, each herein incorporatedby reference, in their entirety, for all purposes.

XI.B.2 Shared Antigen Vaccine Sequence Selection

Shared antigen sequences for inclusion in a shared antigen vaccine andappropriate patients for treatment with such vaccine can be chosen byone of skill in the art using the detailed disclosure provided herein.In certain instances a particular mutation and HLA allele combinationcan be preferred (e.g., based on sequencing data available from a givensubject indicating that each are present in the subject) andsubsequently used in combination together to identify a sharedneoantigen sequence.

XIII. Example Computer

A computer can be used for any of the computational methods describedherein. One skilled in the art will recognize a computer can havedifferent architectures. Examples of computers are known to thoseskilled in the art, for example the computers described in more detailin U.S. Pat. No. 10,055,540, US Application Pub. No. US20200010849A1,and international patent application publications WO/2018/195357 andWO/2018/208856, each herein incorporated by reference, in theirentirety, for all purposes.

XIV. Antigen Delivery Vector Example

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: MackPublishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry3^(rd) Ed. (Plenum Press) Vols A and B(1992).

XIV.A. Neoantigen Cassette Design

Through vaccination, multiple class I MHC restricted tumor-specificneoantigens (TSNAs) that stimulate the corresponding cellular immuneresponse(s) can be delivered. In one example, a vaccine cassette wasengineered to encode multiple epitopes as a single gene product wherethe epitopes were either embedded within their natural, surroundingpeptide sequence or spaced by non-natural linker sequences. Severaldesign parameters were identified that could potentially impact antigenprocessing and presentation and therefore the magnitude and breadth ofthe TSNA specific CD8 T cell responses. In the present example, severalmodel cassettes were designed and constructed to evaluate: (1) whetherrobust T cell responses could be generated to multiple epitopesincorporated in a single expression cassette; (2) what makes an optimallinker placed between the TSNAs within the expression cassette-thatleads to optimal processing and presentation of all epitopes; (3) if therelative position of the epitopes within the cassette impact T cellresponses; (4) whether the number of epitopes within a cassetteinfluences the magnitude or quality of the T cell responses toindividual epitopes; (5) if the addition of cellular targeting sequencesimproves T cell responses.

Two readouts were developed to evaluate antigen presentation and T cellresponses specific for marker epitopes within the model cassettes: (1)an in vitro cell-based screen which allowed assessment of antigenpresentation as gauged by the activation of specially engineeredreporter T cells (Aarnoudse et al., 2002; Nagai et al., 2012); and (2)an in vivo assay that used HLA-A2 transgenic mice (Vitiello et al.,1991) to assess post-vaccination immunogenicity of cassette-derivedepitopes of human origin by their corresponding epitope-specific T cellresponses (Cornet et al., 2006; Depla et al., 2008; Ishioka et al.,1999).

XIV.B. Antigen Cassette Design Evaluation

XIV.B.1. Methods and Materials

TCR and Cassette Design and Cloning

The selected TCRs recognize peptides NLVPMVATV (SEQ ID NO: 78) (PDB#5D2N), CLGGLLTMV (SEQ ID NO: 79) (PDB #3REV), GILGFVFTL (SEQ ID NO: 80)(PDB #1OGA) LLFGYPVYV (SEQ ID NO: 81) (PDB #1A07) when presented byA*0201. Transfer vectors were constructed that contain 2A peptide-linkedTCR subunits (beta followed by alpha), the EMCV IRES, and 2A-linked CD8subunits (beta followed by alpha and by the puromycin resistance gene).Open reading frame sequences were codon-optimized and synthesized byGeneArt.

Cell Line Generation for In Vitro Epitope Processing and PresentationStudies

Peptides were purchased from ProImmune or Genscript diluted to 10 mg/mLwith 10 mM tris(2-carboxyethyl)phosphine (TCEP) in water/DMSO (2:8,v/v). Cell culture medium and supplements, unless otherwise noted, werefrom Gibco. Heat inactivated fetal bovine serum (FBShi) was fromSeradigm. QUANTI-Luc Substrate, Zeocin, and Puromycin were fromInvivoGen. Jurkat-Lucia NFAT Cells (InvivoGen) were maintained in RPMI1640 supplemented with 10% FBShi, Sodium Pyruvate, and 100 μg/mL Zeocin.Once transduced, these cells additionally received 0.3 μg/mL Puromycin.T2 cells (ATCC CRL-1992) were cultured in Iscove's Medium (IMDM) plus20% FBShi. U-87 MG (ATCC HTB-14) cells were maintained in MEM EaglesMedium supplemented with 10% FBShi.

Jurkat-Lucia NFAT cells contain an NFAT-inducible Lucia reporterconstruct. The Lucia gene, when activated by the engagement of the Tcell receptor (TCR), causes secretion of a coelenterazine-utilizingluciferase into the culture medium. This luciferase can be measuredusing the QUANTI-Luc luciferase detection reagent. Jurkat-Lucia cellswere transduced with lentivirus to express antigen-specific TCRs. TheHIV-derived lentivirus transfer vector was obtained from GeneCopoeia,and lentivirus support plasmids expressing VSV-G (pCMV-VsvG), Rev(pRSV-Rev) and Gag-pol (pCgpV) were obtained from Cell Design Labs.

Lentivirus was prepared by transfection of 50-80% confluent T75 flasksof HEK293 cells with Lipofectamine 2000 (Thermo Fisher), using 40 μl oflipofectamine and 20 μg of the DNA mixture (4:2:1:1 by weight of thetransfer plasmid:pCgpV:pRSV-Rev:pCMV-VsvG). 8-10 mL of thevirus-containing media were concentrated using the Lenti-X system(Clontech), and the virus resuspended in 100-200 μl of fresh medium.This volume was used to overlay an equal volume of Jurkat-Lucia cells(5×10E4-1×10E6 cells were used in different experiments). Followingculture in 0.3 μg/ml puromycin-containing medium, cells were sorted toobtain clonality. These Jurkat-Lucia TCR clones were tested for activityand selectivity using peptide loaded T2 cells.

In Vitro Epitope Processing and Presentation Assay

T2 cells are routinely used to examine antigen recognition by TCRs. T2cells lack a peptide transporter for antigen processing (TAP deficient)and cannot load endogenous peptides in the endoplasmic reticulum forpresentation on the MHC. However, the T2 cells can easily be loaded withexogenous peptides. The five marker peptides (NLVPMVATV (SEQ ID NO: 78),CLGGLLTMV (SEQ ID NO: 79), GLCTLVAML (SEQ ID NO: 82), LLFGYPVYV (SEQ IDNO: 81), GILGFVFTL (SEQ ID NO: 80)) and two irrelevant peptides(WLSLLVPFV (SEQ ID NO: 83), FLLTRICT (SEQ ID NO: 84)) were loaded ontoT2 cells. Briefly, T2 cells were counted and diluted to 1×106 cells/mLwith IMDM plus 1% FBShi. Peptides were added to result in 10 μgpeptide/1×106 cells. Cells were then incubated at 37° C. for 90 minutes.Cells were washed twice with IMDM plus 20% FBShi, diluted to 5×10E5cells/mL and 100 μL plated into a 96-well Costar tissue culture plate.Jurkat-Lucia TCR clones were counted and diluted to 5×10E5 cells/mL inRPMI 1640 plus 10% FBShi and 100 μL added to the T2 cells. Plates wereincubated overnight at 37° C., 5% CO2. Plates were then centrifuged at400 g for 3 minutes and 20 μL supernatant removed to a white flat bottomGreiner plate. QUANTI-Luc substrate was prepared according toinstructions and 50 μL/well added. Luciferase expression was read on aMolecular Devices SpectraMax iE3x.

To test marker epitope presentation by the adenoviral cassettes, U-87 MGcells were used as surrogate antigen presenting cells (APCs) and weretransduced with the adenoviral vectors. U-87 MG cells were harvested andplated in culture media as 5×10E5 cells/100 μl in a 96-well Costartissue culture plate. Plates were incubated for approximately 2 hours at37° C. Adenoviral cassettes were diluted with MEM plus 10% FBShi to anMOI of 100, 50, 10, 5, 1 and 0 and added to the U-87 MG cells as 5μl/well. Plates were again incubated for approximately 2 hours at 37° C.Jurkat-Lucia TCR clones were counted and diluted to 5×10E5 cells/mL inRPMI plus 10% FBShi and added to the U-87 MG cells as 100 μL/well.Plates were then incubated for approximately 24 hours at 37° C., 5% CO2.Plates were centrifuged at 400 g for 3 minutes and 20 μL supernatantremoved to a white flat bottom Greiner plate. QUANTI-Luc substrate wasprepared according to instructions and 50 μL/well added. Luciferaseexpression was read on a Molecular Devices SpectraMax iE3x.

Mouse Strains for Immunogenicity Studies

Transgenic HLA-A2.1 (HLA-A2 Tg) mice were obtained from Taconic Labs,Inc. These mice carry a transgene consisting of a chimeric class Imolecule comprised of the human HLA-A2.1 leader, α1, and α2 domains andthe murine H2-Kb α3, transmembrane, and cytoplasmic domains (Vitiello etal., 1991). Mice used for these studies were the first generationoffspring (F1) of wild type BALB/cAnNTac females and homozygous HLA-A2.1Tg males on the C57Bl/6 background.

Adenovirus Vector (Ad5v) Immunizations

HLA-A2 Tg mice were immunized with 1×10¹⁰ to 1×10⁶ viral particles ofadenoviral vectors via bilateral intramuscular injection into thetibialis anterior. Immune responses were measured at 12 dayspost-immunization.

Lymphocyte Isolation

Lymphocytes were isolated from freshly harvested spleens and lymph nodesof immunized mice. Tissues were dissociated in RPMI containing 10% fetalbovine serum with penicillin and streptomycin (complete RPMI) using theGentleMACS tissue dissociator according to the manufacturer'sinstructions.

Ex Vivo Enzyme-Linked Immunospot (ELISPOT) Analysis

ELISPOT analysis was performed according to ELISPOT harmonizationguidelines (Janetzki et al., 2015) with the mouse IFNg ELISpotPLUS kit(MABTECH). 1×10⁵ splenocytes were incubated with 10 uM of the indicatedpeptides for 16 hours in 96-well IFNg antibody coated plates. Spots weredeveloped using alkaline phosphatase. The reaction was timed for 10minutes and was quenched by running the plate under tap water. Spotswere counted using an AID vSpot Reader Spectrum. For ELISPOT analysis,wells with saturation >50% were recorded as “too numerous to count”.Samples with deviation of replicate wells >10% were excluded fromanalysis. Spot counts were then corrected for well confluency using theformula: spot count+2×(spot count×% confluence/[100%−% confluence]).Negative background was corrected by subtraction of spot counts in thenegative peptide stimulation wells from the antigen stimulated wells.Finally, wells labeled too numerous to count were set to the highestobserved corrected value, rounded up to the nearest hundred.

Ex Vivo Intracellular Cytokine Staining (ICS) and Flow CytometryAnalysis

Freshly isolated lymphocytes at a density of 2-5×10⁶ cells/mL wereincubated with 10 uM of the indicated peptides for 2 hours. After twohours, brefeldin A was added to a concentration of 5 ug/ml and cellswere incubated with stimulant for an additional 4 hours. Followingstimulation, viable cells were labeled with fixable viability dyeeFluor780 according to manufacturer's protocol and stained with anti-CD8APC (clone 53-6.7, BioLegend) at 1:400 dilution. Anti-IFNg PE (cloneXMG1.2, BioLegend) was used at 1:100 for intracellular staining. Sampleswere collected on an Attune NxT Flow Cytometer (Thermo Scientific). Flowcytometry data was plotted and analyzed using FlowJo. To assess degreeof antigen-specific response, both the percent IFNg+ of CD8+ cells andthe total IFNg+ cell number/1×10⁶ live cells were calculated in responseto each peptide stimulant.

XIV.B.2. In Vitro Evaluation of Antigen Cassette Designs

As an example of antigen cassette design evaluation, an in vitrocell-based assay was developed to assess whether selected human epitopeswithin model vaccine cassettes were being expressed, processed, andpresented by antigen-presenting cells (FIG. 1 ). Upon recognition,Jurkat-Lucia reporter T cells that were engineered to express one offive TCRs specific for well-characterized peptide-HLA combinationsbecome activated and translocate the nuclear factor of activated T cells(NFAT) into the nucleus which leads to transcriptional activation of aluciferase reporter gene. Antigenic stimulation of the individualreporter CD8 T cell lines was quantified by bioluminescence.

Individual Jurkat-Lucia reporter lines were modified by lentiviraltransduction with an expression construct that includes anantigen-specific TCR beta and TCR alpha chain separated by a P2Aribosomal skip sequence to ensure equimolar amounts of translatedproduct (Banu et al., 2014). The addition of a second CD8 beta-P2A-CD8alpha element to the lentiviral construct provided expression of the CD8co-receptor, which the parent reporter cell line lacks, as CD8 on thecell surface is crucial for the binding affinity to target pMHCmolecules and enhances signaling through engagement of its cytoplasmictail (Lyons et al., 2006; Yachi et al., 2006).

After lentiviral transduction, the Jurkat-Lucia reporters were expandedunder puromycin selection, subjected to single cell fluorescenceassisted cell sorting (FACS), and the monoclonal populations tested forluciferase expression. This yielded stably transduced reporter celllines for specific peptide antigens 1, 2, 4, and 5 with functional cellresponses. (Table 2).

TABLE 2 Development of an in vitro T cell activation assay.Peptide-specific T cell recognition as measured by induction ofluciferase indicates effective processing and presentation of thevaccine cassette antigens. Short Cassette Design Epitope AAY 1 24.5 ±0.5 2 11.3 ± 0.4 3* n/a 4 26.1 ± 3.1 5 46.3 ± 1.9 *Reporter T cell forepitope 3 not yet generated

In another example, a series of short cassettes, all marker epitopeswere incorporated in the same position (FIG. 2A) and only the linkersseparating the HLA-A*0201 restricted epitopes (FIG. 2B) were varied.Reporter T cells were individually mixed with U-87 antigen-presentingcells (APCs) that were infected with adenoviral constructs expressingthese short cassettes, and luciferase expression was measured relativeto uninfected controls. All four antigens in the model cassettes wererecognized by matching reporter T cells, demonstrating efficientprocessing and presentation of multiple antigens. The magnitude of Tcell responses follow largely similar trends for the natural andAAY-linkers. The antigens released from the RR-linker based cassetteshow lower luciferase inductions (Table 3). The DPP-linker, designed todisrupt antigen processing, produced a vaccine cassette that led to lowepitope presentation (Table 3).

TABLE 3 Evaluation of linker sequences in short cassettes. Luciferaseinduction in the in vitro T cell activation assay indicated that, apartfrom the DPP-based cassette, all linkers facilitated efficient releaseof the cassette antigens. T cell epitope only (no linker) = 9AA, naturallinker one side = 17AA, natural linker both sides = 25AA, non-naturallinkers = AAY, RR, DPP Short Cassette Designs Epitope 9AA 17AA 25AA AAYRR DPP 1 33.6 ± 0.9 42.8 ± 2.1 42.3 ± 2.3 24.5 ± 0.5 21.7 ± 0.9 0.9 ±0.1 2 12.0 ± 0.9 10.3 ± 0.6 14.6 ± 04  11.3 ± 0.4  8.5 ± 0.3 1.1 ± 0.23* n/a n/a n/a n/a n/a n/a 4 26.6 ± 2.5 16.1 ± 0.6 16.6 ± 0.8 26.1 ± 3.112.5 ± 0.8 1.3 ± 0.2 5 29.7 ± 0.6 21.2 ± 0.7 24.3 ± 1.4 46.3 ± 1.9 19.7± 0.4 1.3 ± 0.1 *Reporter T cell for epitope 3 not yet generated

In another example, an additional series of short cassettes wereconstructed that, besides human and mouse epitopes, contained targetingsequences such as ubiquitin (Ub), MHC and Ig-kappa signal peptides (SP),and/or MHC transmembrane (TM) motifs positioned on either the N- orC-terminus of the cassette. (FIG. 3 ). When delivered to U-87 APCs byadenoviral vector, the reporter T cells again demonstrated efficientprocessing and presentation of multiple cassette-derived antigens.However, the magnitude of T cell responses were not substantiallyimpacted by the various targeting features (Table 4).

TABLE 4 Evaluation of cellular targeting sequences added to modelvaccine cassettes. Employing the in vitro T cell activation assaydemonstrated that the four HLA-A*0201 restricted marker epitopes areliberated efficiently from the model cassettes and targeting sequencesdid not substantially improve T cell recognition and activation. ShortCassette Designs Epitope A B C D E F G H I J 1 32.5 ± 1.5 31.8 ± 0.829.1 ± 1.2 29.1 ± 1.1 28.4 ± 0.7 20.4 ± 0.5 35.0 ± 1.3 30.3 ± 2.0 22.5 ±0.9 38.1 ± 1.6 2  6.1 ± 0.2  6.3 ± 0.2  7.6 ± 0.4  7.0 ± 0.5  5.9 ± 0.2 3.7 ± 0.2  7.6 ± 0.4  5.4 ± 0.3  6.2 ± 0.4  6.4 ± 0.3 3* n/a n/a n/an/a n/a n/a n/a n/a n/a n/a 4 12.3 ± 1.1 14.1 ± 0.7 12.2 ± 0.8 13.7 ±1.0 11.7 ± 0.8 10.6 ± 0.4 11.0 ± 0.6  7.6 ± 0.6 16.1 ± 0.5  8.7 ± 0.5 544.4 ± 2.8 53.6 ± 1.6 49.9 ± 3.3 50.5 ± 2.8 41.7 ± 2.8 36.1 ± 1.1 46.5 ±2.1 31.4 ± 0.6 75.4 ± 1.6 35.7 ± 2.2 *Reporter T cell for epitope 3 notyet generated

XIV.B.3. In Vivo Evaluation of Antigen Cassette Designs

As another example of antigen cassette design evaluation, vaccinecassettes were designed to contain 5 well-characterized human class IMHC epitopes known to stimulate CD8 T cells in an HLA-A*02:01 restrictedfashion (FIG. 2A, 3, 5A). For the evaluation of their in vivoimmunogenicity, vaccine cassettes containing these marker epitopes wereincorporated in adenoviral vectors and used to infect HLA-A2 transgenicmice (FIG. 4 ). This mouse model carries a transgene consisting partlyof human HLA-A*0201 and mouse H2-Kb thus encoding a chimeric class I MHCmolecule consisting of the human HLA-A2.1 leader, α1 and α2 domainsligated to the murine 3, transmembrane and cytoplasmic H2-Kb domain(Vitiello et al., 1991). The chimeric molecule allowsHLA-A*02:01-restricted antigen presentation whilst maintaining thespecies-matched interaction of the CD8 co-receptor with the α3 domain onthe MHC.

For the short cassettes, all marker epitopes generated a T cellresponse, as determined by IFN-gamma ELISPOT, that was approximately10-50× stronger of what has been commonly reported (Cornet et al., 2006;Depla et al., 2008; Ishioka et al., 1999). Of all the linkers evaluated,the concatamer of 25mer sequences, each containing a minimal epitopeflanked by their natural amino acids sequences, generated the largestand broadest T cell response (Table 5). Intracellular cytokine staining(ICS) and flow cytometry analysis revealed that the antigen-specific Tcell responses are derived from CD8 T cells.

TABLE 5 In vivo evaluation of linker sequences in short cassettes.ELISPOT data indicated that HLA-A2 transgenic mice, 17 dayspost-infection with 1e11 adenovirus viral particles, generated a T cellresponse to all class I MHC restricted epitopes in the cassette. ShortCassette Designs Epitope 9AA 17AA 25AA AAY RR DPP 1 2020 +/− 583  2505+/− 1281 6844 +/− 956 1489 +/− 762  1675 +/− 690  1781 +/− 774  2 4472+/− 755  3792 +/− 1319 7629 +/− 996 3851 +/− 1748 4726 +/− 1715 5868 +/−1427 3 5830 +/− 315 3629 +/− 862 7253 +/− 491 4813 +/− 1761 6779 +/−1033 7328 +/− 1700 4 5536 +/− 375 2446 +/− 955  2961 +/− 1487 4230 +/−1759 6518 +/− 909  7222 +/− 1824 5 8800 +/− 0  7943 +/− 821 8423 +/− 4428312 +/− 696  8800 +/− 0   1836 +/− 328 

In another example, a series of long vaccine cassettes was constructedand incorporated in adenoviral vectors that, next to the original 5marker epitopes, contained an additional 16 HLA-A*02:01, A*03:01 andB*44:05 epitopes with known CD8 T cell reactivity (FIG. 5A, 5B). Thesize of these long cassettes closely mimicked the final clinicalcassette design, and only the position of the epitopes relative to eachother was varied. The CD8 T cell responses were comparable in magnitudeand breadth for both long and short vaccine cassettes, demonstratingthat (a) the addition of more epitopes did not substantially impact themagnitude of immune response to the original set of epitopes, and (b)the position of an epitope in a cassette did not substantially influencethe ensuing T cell response to it (Table 6).

TABLE 6 In vivo evaluation of the impact of epitope position in longcassettes. ELISPOT data indicated that HLA-A2 transgenic mice, 17 dayspost-infection with 5e10 adenovirus viral particles, generated a T cellresponse comparable in magnitude for both long and short vaccinecassettes. Long Cassette Designs Epitope Standard Scrambled Short 1  863+/− 1080  804 +/− 1113 1871 +/− 2859 2 6425 +/− 1594 28 +/− 62 5390 +/−1357 3* 23 +/− 30 36 +/− 18  0 +/− 48 4 2224 +/− 1074 2727 +/− 644  2637+/− 1673 5 7952 +/− 297  8100 +/− 0    8100 +/− 0    *Suspectedtechnical error caused an absence of a T cell response.

XIV.B.4. Antigen Cassette Design for Immunogenicity and ToxicologyStudies

In summary, the findings of the model cassette evaluations (FIG. 2-5 ,Tables 2-6) demonstrated that, for model vaccine cassettes, robustimmunogenicity was achieved when a “string of beads” approach wasemployed that encodes around 20 epitopes in the context of anadenovirus-based vector. The epitopes were assembled by concatenating25mer sequences, each embedding a minimal CD8 T cell epitope (e.g. 9amino acid residues) that were flanked on both sides by its natural,surrounding peptide sequence (e.g. 8 amino acid residues on each side).As used herein, a “natural” or “native” flanking sequence refers to theN- and/or C-terminal flanking sequence of a given epitope in thenaturally occurring context of that epitope within its source protein.For example, the HCMV pp65 MHC I epitope NLVPMVATV (SEQ ID NO: 78) isflanked on its 5′ end by the native 5′ sequence WQAGILAR (SEQ ID NO: 85)and on its 3′ end by the native 3′ sequence QGQNLKYQ (SEQ ID NO: 86),thus generating the WQAGILARNLVPMVATVQGQNLKYQ (SEQ ID NO: 87) 25merpeptide found within the HCMV pp65 source protein. The natural or nativesequence can also refer to a nucleic acid sequence that encodes anepitope flanked by native flanking sequence(s). Each 25mer sequence isdirectly connected to the following 25mer sequence. In instances wherethe minimal CD8 T cell epitope is greater than or less than 9 aminoacids, the flanking peptide length can be adjusted such that the totallength is still a 25mer peptide sequence. For example, a 10 amino acidCD8 T cell epitope can be flanked by an 8 amino acid sequence and a 7amino acid. The concatamer was followed by two universal class II MHCepitopes that were included to stimulate CD4 T helper cells and improveoverall in vivo immunogenicity of the vaccine cassette antigens.(Alexander et al., 1994; Panina-Bordignon et al., 1989) The class IIepitopes were linked to the final class I epitope by a GPGPG amino acidlinker (SEQ ID NO:56). The two class II epitopes were also linked toeach other by a GPGPG amino acid linker (SEQ ID NO: 56), as a well asflanked on the C-terminus by a GPGPG amino acid linker (SEQ ID NO: 56).Neither the position nor the number of epitopes appeared tosubstantially impact T cell recognition or response. Targeting sequencesalso did not appear to substantially impact the immunogenicity ofcassette-derived antigens.

As a further example, based on the in vitro and in vivo data obtainedwith model cassettes (FIG. 2-5 , Tables 2-6), a cassette design wasgenerated that alternates well-characterized T cell epitopes known to beimmunogenic in nonhuman primates (NHPs), mice and humans. The 20epitopes, all embedded in their natural 25mer sequences, are followed bythe two universal class II MHC epitopes that were present in all modelcassettes evaluated (FIG. 6 ). This cassette design was used to studyimmunogenicity as well as pharmacology and toxicology studies inmultiple species.

XIV.B.5. Antigen Cassette Design and Evaluation for 30, 40, and 50Antigens

Large antigen cassettes were designed that had either 30 (L), 40 (XL) or50 (XXL) epitopes, each 25 amino acids in length. The epitopes were amix of human, NHP and mouse epitopes to model disease antigens includingtumor antigens. FIG. 29 illustrates the general organization of theepitopes from the various species. The model antigens used are describedin Tables 32, 33 and 34 for human, primate, and mouse model epitopes,respectively. Each of Tables 32, 33 and 34 described the epitopeposition, name, minimal epitope description, and MHC class.

These cassettes were cloned into the ChAdV68 and alphavirus vaccinevectors as described to evaluate the efficacy of longer multiple-epitopecassettes. FIG. 30 shows that each of the large antigen cassettes wereexpressed from a ChAdV vector as indicated by at least one major band ofthe expected size by Western blot.

Mice were immunized as described to evaluate the efficacy of the largecassettes. T cell responses were analyzed by ICS and tetramer stainingfollowing immunization with a ChAdV68 vector (FIG. 31 /Table 35 and FIG.32 /Table 36, respectively) and by ICS following immunization with asrRNA vector (FIG. 33 /Table 37) for epitopes AH1 (top panels) andSIINFEKL (SEQ ID NO: 72) (bottom panels). Immunizations using ChAdV68and srRNA vaccine vectors expressing either 30 (L), 40 (XL) or 50 (XXL)epitopes induced CD8+ immune responses to model disease epitopes.

TABLE 32 Human epitopes in large cassettes (TABLE 32 discloses SEQ ID NOS 80-82, 78-79, 88-100, 87and 101-111, respectively, in order of columns) Epitope positionin each cassette L XL XXL Name Minimal epitope 25 mer MHC RestrictionStrain Species 3 3 3 5.influenza M GILGFVFTL PILSPLTKGILGFVFTLTVPSERGLClass I A*02:01 Human Human 6 6 6 4.HTLV-1 Tax LLFGYPVYVHFPGFGQSLLFGYPVYVFGDCVQGD Class I A*02:01 Human Human 9 9 9 3.EBV BMLF1GLCTLVAML RMQAIQNAGLCTLVAMLEETIFWLQ Class I A*02:01 Human Human 12 12 121.HCMV pp65 NLVPMVATV WQAGILARNLVPMVATVQGQNLKYQ Class I A*02:01 HumanHuman 15 15 15 2.EBV LMP2A CLGGLLTMV RTYGPVFMCLGGLLTMVAGAVWLTV Class IA*02:01 Human Human 18 18 18 CT83 NTDNNLAVY SSSGLINSNTDNNLAVYDLSRDILNClass I A*01:01 Human Human 21 21 MAGEA6 EVDPIGHVYLVEGIELMEVDPIGHVYIFATCLGL Class I B*35:01 Human Human 21 25 25 CT83LLASSILCA MNFYLLLASSILCALIVFWKYRRFQ Class I A*02:01 Human Human 24 31 28FOXE1 AIFPGAVPAA AAAAAAAAIFPGAVPAARPPYPGAV Class I A*02:01 Human Human27 35 32 CT83 VYDLSRDIL SNTDNNLAVYDLSRDILNNFPHSIA Class I A*24:02 HumanHuman 38 36 MAGE3/6 ASSLPTTMNY DPPQSPQGASSLPTTMNYPLWSQSY Class I A*01:01Human Human 30 40 40 Influenza HA PKYVKQNTLKLATITYGACPKYVKQNTLKLATGMRNVP Class II DRB1*0101 Human Human 44 CMV pp65LPLKMLNIPSINVH SIYVYALPLKMLNIPSINVHHYPSA Class II DRB1*0101 Human Human47 EBV EBNA3A PEQWMFQGAPPSQGT EGPWVPEQWMFQGAPPSQGTDVVQH Class IIDRB1*0102 Human Human 50 CMV pp65 EHPTFTSQYRIQGKLRGPQYSEHPTFTSQYRIQGKLEYRH Class II DRB1*1101 Human Human

TABLE 33 NHP epitopes in large cassettes (TABLE 33 discloses SEQ ID NOS 112-141,respectively, in order of columns) Epitope position in each cassetteepitope L XL XXL Name Minimal  25 mer MHC Restriction Strain Species 1 11 Gag CM9 CTPYDINQM MFQALSEGCTPYD Class I Mamu-A*01  Rhesus NHPINQMLNVLGDHQ 4 4 4 Tat TL8 TTPESANL SCISEADATTPES Class I Mamu-A*01 Rhesus NHP ANLGEEILSQLY 7 7 7 Env CL9 CAPPGYALL WDAIRFRYCAPPG Class IMamu-A*01  Rhesus NHP YALLRCNDTNYS 10 10 10 Pol SV9 SGPKTNIIVAFLMALTDSGPKT Class I Mamu-A*01  Rhesus NHP NIIVDSQYVMGI 13 13 13Gag LW9 LSPRTLNAW GNVWVHTPLSPRT Class I Mamu-A*01  Rhesus NHPLNAWVKAVEEKK 16 Env_TL9 TVPWPNASL AFRQVCHTTVPWP Class I Mamu-A*01 Rhesus NHP NASLTPKWNNET 16 16 19 Ag856 PNGTHSWEYWGAQLN VFNFPPNGTHSWEClass II Mamu-DR*W  Rhesus NHP YWGAQLNAMKGD 19 19 23 HIV-1 EnvYKYKVVKIEPLGV NWRSELYKYKVVK Class II Mamu-DR*W  Rhesus NHP IEPLGVAPTKAK26 Gag TE15 TEEAKQIVQRHLV EKVKHTEEAKQIV Class II Mamu-DRB*  Rhesus NHPVE QRHLVVETGTTE 23 30 CFP-10  AGSLQGQWRGAAG DQVESTAGSLQGQ Class IIMafa-DRB1* Cyno NHP 36-48 WRGAAGTAAQAA 27 34 CFP-10  EISTNIRQAGVQYQELDEISTNIRQA Class II Mafa-DRB1* Cyno NHP 71-86 SRA GVQYSRADEEQQ 22 2938 Env 338-346 RPKQAWCWF FHSQPINERPKQA Class I Mafa-  Cyno NHPWCWEGGSWKEAI A1*063 25 33 42 Nef 103-111 RPKVPLRTM DDIDEEDDDLVGV Class IMafa-  Cyno NHP SVRPKVPLRTMS A1*063 28 37 45 Gag 386-394 GPRKPIKCWPFAAAQQRGPRKP Class I Mafa-  Cyno NHP IKCWNCGKEGHS A1*063 48 Nef LT9LNMADKKET RRLTARGLLNMAD Class I Mafa-  Cyno NHP KKETRTPKKAKA B*1043

TABLE 34Mouse epitopes in large cassettes (TABLE 34 discloses SEQ ID NOS 72, 142-144,73, 145-161, 75-76 and 162-177, respectively, in order of columns)Epitopes in large cassettes Minimal  L XL XXL Name epitope 25 mer MHCRestriction Strain Species 2 2 2 OVA257 SIINFEKL VSGLEQLESIINF Class IH2-Kb B6 Mouse EKLTEWTSSNVM 5 B16-EGP EGPRNQDWL ALLAVGALEGPRN Class IH2-Db B6 Mouse QDWLGVPRQLVT 8 B16-TRP1 455-463 TAPDNLGYM VTNTEMFVTAPDNClass I H2-Db B6 Mouse LGYMYEVQWPGQ 11 Trp2180-188 SVYDFFVWLTQPQIANCSVYDF Class I H2-Kb B6 Mouse FVWLHYYSVRDT 5 5 14 CT26 AH1-A5SPSYAYHQF LWPRVTYHSPSYA Class I H2-Ld Balb/C Mouse YHQFERRAKYKR 8 17CT26 AH1-39 MNKYAYHML LWPRVTYHMNKYA Class I H2-Ld Balb/C MouseYHMLERRAKYKR 11 20 MC38 Dpagt1 SIIVFNLL GQSLVISASIIVF Class I H2-Kb B6Mouse NLLELEGDYRDD 14 22 MC38 Adpgk ASMTNMELM GIPVHLELASMTN Class IH2-Db B6 Mouse MELMSSIVHQQV 17 24 MC38 Reps1 AQLANDVVL RVLELFRAAQLANClass I H2-Db B6 Mouse DVVLQIMELCGA 8 20 27 P815 P1A 35-44 LPYLGWLVFHRYSLEEILPYLG Class I H2-Ld DBA/2 Mouse WLVFAVVTTSFL 11 22 29 P815 P1EGYCGLRGTGV YLSKNPDGYCGLR Class I H2-Kd DBA/2 Mouse GTGVSCPMAIKK 14 24 31Panc02 Mesothelin LSIFKHKL NEIPFTYEQLSIF Class I H2-Kb B6 MouseKHKLDKTYPQGY 17 26 33 Panc02 Mesothelin LIWIPALL SRASLLGPGFVLI Class IH2-Kb B6 Mouse WIPALLPALRLS 20 28 35 ID8 FRa 161-169 SSGHNECPVNWHKGWNWSSGHN Class I H2-Kb B6 Mouse ECPVGASCHPFT 23 30 37ID8 Mesothelin 400 GQKMNAQAI KTLLKVSKGQKMN Class I H2-Db B6 MouseAQAIALVACYLR 26 32 39 OVA-II ISQAVHAAH ESLKISQAVHAAH Class II I-Ab, I-AdB6 Mouse AEINEAGR AEINEAGREVVG 29 34 41 ESAT-6 MTEQQWNFAG MTEQQWNFAGIEAClass II I-Ab B6 Mouse IEAAASAIQ AASAIQGNVTSI 36 43 TT p30 FNNFTVSFWLDMFNNFTVSFWLR Class II I-Ad Balb/C Mouse RVPKVSASHL VPKVSASHLEQY 39 46HEL DGSTDYGILQ TNRNTDGSTDYGI Class II I-Ak CBA Mouse INSRW LQINSRWWCNDG49 MOG MEVGWYRSPF TGMEVGWYRSPFS Class II I-Ab B6 Mouse SRVVHLYRNRVVHLYRNGKDQ

TABLE 35 Average IFNg+ cells in response to AH1 andSIINFEKL (SEQ ID NO: 72) peptides inChAd large cassette treated mice. Data ispresented as % of total CD8 cells. Shown isaverage and standard deviation per group andp-value by ANOVA with Tukey's test. Allp-valuescompared to MAG 20-antigen cassette. # Standard antigens AntigenAverage deviation p-value N 20 SIINFEKL 5.308 0.660 n/a 8 30 SIINFEKL4.119 1.019 0.978 8 40 SIINFEKL 6.324 0.954 0.986 8 50 SIINFEKL 8.1691.469 0.751 8 20 AH1 6.405 2.664 n/a 8 30 AH1 4.373 1.442 0.093 8 40 AH14.126 1.135 0.050 8 50 AH1 4.216 0.808 0.063 8

TABLE 36 Average tetramer+ cells for AH1 and SIINFEKLantigens in ChAd large cassette treated mice.Data is presented as % of total CD8 cells.Shown is average and standard deviationper group and p-value by ANOVA with Tukey'stest. All p-values compared to MAG 20-  antigen cassette. # Standardantigens Antigen Average deviation p-value N 20 SIINFEKL 10.314 2.384n/a 8 30 SIINFEKL 4.551 2.370 0.003 8 40 SIINFEKL 5.186 3.254 0.009 8 50SIINFEKL 14.113 3.660 0.072 8 20 AH1 6.864 2.207 n/a 8 30 AH1 4.7130.922 0.036 8 40 AH1 5.393 1.452 0.223 8 50 AH1 5.860 1.041 0.543 8

TABLE 37 Average IFNg+ cells in response to AH1 andSIINFEKL peptides in SAM large cassettetreated mice. Data is presented as % of totalCD8 cells. Shown is average and standarddeviation per group and p-value by ANOVAwith Tukey's test. All p-values compared to MAG 20-antigen cassette. #Standard antigens Antigen Average deviation p-value N 20 SIINFEKL 1.8430.422 n/a 8 30 SIINFEKL 2.112 0.522 0.879 7 40 SIINFEKL 1.754 0.9780.995 7 50 SIINFEKL 1.409 0.766 0.606 8 20 AH1 3.050 0.909 n/a 8 30 AH10.618 0.427 1.91E−05 7 40 AH1 1.286 0.284 0.001 7 50 AH1 1.309 1.1490.001 8

XV. ChAd Antigen Cassette Delivery Vector

XV.A. ChAd Antigen Cassette Delivery Vector Construction

In one example, Chimpanzee adenovirus (ChAd) was engineered to be adelivery vector for antigen cassettes. In a further example, afull-length ChAdV68 vector was synthesized based on AC_000011.1(sequence 2 from U.S. Pat. No. 6,083,716) with E1 (nt 457 to 3014) andE3 (nt 27,816-31,332) sequences deleted. Reporter genes under thecontrol of the CMV promoter/enhancer were inserted in place of thedeleted E1 sequences. Transfection of this clone into HEK293 cells didnot yield infectious virus. To confirm the sequence of the wild-type C68virus, isolate VR-594 was obtained from the ATCC, passaged, and thenindependently sequenced (SEQ ID NO:10). When comparing the AC_000011.1sequence to the ATCC VR-594 sequence (SEQ ID NO:10) of wild-type ChAdV68virus, 6 nucleotide differences were identified. In one example, amodified ChAdV68 vector was generated based on AC_000011.1, with thecorresponding ATCC VR-594 nucleotides substituted at five positions(ChAdV68.5WTnt SEQ ID NO:1).

In another example, a modified ChAdV68 vector was generated based onAC_000011.1 with E1 (nt 577 to 3403) and E3 (nt 27,816-31,332) sequencesdeleted and the corresponding ATCC VR-594 nucleotides substituted atfour positions. A GFP reporter (ChAdV68.4WTnt.GFP; SEQ ID NO:11) ormodel neoantigen cassette (ChAdV68.4WTnt.MAG25mer; SEQ ID NO:12) underthe control of the CMV promoter/enhancer was inserted in place ofdeleted E1 sequences.

In another example, a modified ChAdV68 vector was generated based onAC_000011.1 with E1 (nt 577 to 3403) and E3 (nt 27,125-31,825) sequencesdeleted and the corresponding ATCC VR-594 nucleotides substituted atfive positions. A GFP reporter (ChAdV68.5WTnt.GFP; SEQ ID NO:13) ormodel neoantigen cassette (ChAdV68.5WTnt.MAG25mer; SEQ ID NO:2) underthe control of the CMV promoter/enhancer was inserted in place ofdeleted E1 sequences.

Relevant vectors are described below:

-   -   Full-Length ChAdVC68 sequence “ChAdV68.5WTnt” (SEQ ID NO:1);        AC_000011.1 sequence with corresponding ATCC VR-594 nucleotides        substituted at five positions.    -   ATCC VR-594 C68 (SEQ ID NO:10); Independently sequenced;        Full-Length C68    -   ChAdV684WTnt.GFP (SEQ ID NO:11); AC_000011.1 with E1 (nt 577        to 3403) and E3 (nt 27,816-31,332) sequences deleted;        corresponding ATCC VR-594 nucleotides substituted at four        positions; GFP reporter under the control of the CMV        promoter/enhancer inserted in place of deleted E1    -   ChAdV68.WTnt.MAG25mer (SEQ ID NO:12); AC_000011.1 with E1 (nt        570 to 3403) and E3 (nt 27,816-31,332) sequences deleted;        corresponding ATCC VR-594 nucleotides substituted at four        positions; model neoantigen cassette under the control of the        CMV promoter/enhancer inserted in place of deleted E1    -   CbAdV68.5WTnt.GFP (SEQ ID NO: 13); AC_000011.1 with E1 (nt 577        to 3403) and E3 (nt 27,125-31,825) sequences deleted;        corresponding ATCC VR-594 nucleotides substituted at five        positions; GFP reporter under the control of the CMV        promoter/enhancer inserted in place of deleted E1

XV.B. ChAd Antigen Cassette Delivery Vector Testing

XV.B.1. ChAd Vector Evaluation Methods and Materials

Transfection of HEK293A Cells Using Lipofectamine

DNA for the ChAdV68 constructs (ChAdV68.4WTnt.GFP, ChAdV68.5WTnt.GFP,ChAdV68.4WTnt.MAG25mer and ChAdV68.5WTnt.MAG25mer) was prepared andtransfected into HEK293A cells using the following protocol.

10 ug of plasmid DNA was digested with PacI to liberate the viralgenome. DNA was then purified using GeneJet DNA cleanup Micro columns(Thermo Fisher) according to manufacturer's instructions for long DNAfragments, and eluted in 20 ul of pre-heated water; columns were left at37 degrees for 0.5-1 hours before the elution step.

HEK293A cells were introduced into 6-well plates at a cell density of10⁶ cells/well 14-18 hours prior to transfection. Cells were overlaidwith 1 ml of fresh medium (DMEM-10% hiFBS with pen/strep and glutamate)per well. 1-2 ug of purified DNA was used per well in a transfectionwith twice the ul volume (2-4 ul) of Lipofectamine2000, according to themanufacturer's protocol. 0.5 ml of OPTI-MEM medium containing thetransfection mix was added to the 1 ml of normal growth medium in eachwell, and left on cells overnight.

Transfected cell cultures were incubated at 37° C. for at least 5-7days. If viral plaques were not visible by day 7 post-transfection,cells were split 1:4 or 1:6, and incubated at 37° C. to monitor forplaque development. Alternatively, transfected cells were harvested andsubjected to 3 cycles of freezing and thawing and the cell lysates wereused to infect HEK293A cells and the cells were incubated until virusplaques were observed.

Transfection of ChAdV68 Vectors into HEK293A Cells Using CalciumPhosphate and Generation of the Tertiary Viral Stock

DNA for the ChAdV68 constructs (ChAdV68.4WTnt.GFP, ChAdV68.5WTnt.GFP,ChAdV68.4WTnt.MAG25mer, ChAdV68.5WTnt.MAG25mer) was prepared andtransfected into HEK293A cells using the following protocol.

HEK293A cells were seeded one day prior to the transfection at 10⁶cells/well of a 6 well plate in 5% BS/DMEM/1XP/S, 1×Glutamax. Two wellsare needed per transfection. Two to four hours prior to transfection themedia was changed to fresh media. The ChAdV68.4WTnt.GFP plasmid waslinearized with PacI. The linearized DNA was then phenol chloroformextracted and precipitated using one tenth volume of 3M Sodium acetatepH 5.3 and two volumes of 100% ethanol. The precipitated DNA waspelleted by centrifugation at 12,000×g for 5 min before washing 1× with70% ethanol. The pellet was air dried and re-suspended in 50 μL ofsterile water. The DNA concentration was determined using a NanoDrop™(ThermoFisher) and the volume adjusted to 5 μg of DNA/50 μL.

169 μL of sterile water was added to a microfuge tube. 5 μL of 2M CaCl₂was then added to the water and mixed gently by pipetting. 50 μL of DNAwas added dropwise to the CaCl₂) water solution. Twenty six μL of 2MCaCl₂) was then added and mixed gently by pipetting twice with amicro-pipettor. This final solution should consist of 5 μg of DNA in 250μL of 0.25M CaCl₂). A second tube was then prepared containing 250 μL of2×HBS (Hepes buffered solution). Using a 2 mL sterile pipette attachedto a Pipet-Aid air was slowly bubbled through the 2×HBS solution. At thesame time the DNA solution in the 0.25M CaCl₂ solution was added in adropwise fashion. Bubbling was continued for approximately 5 secondsafter addition of the final DNA droplet. The solution was then incubatedat room temperature for up to 20 minutes before adding to 293A cells.250 μL of the DNA/Calcium phosphate solution was added dropwise to amonolayer of 293A cells that had been seeded one day prior at 10⁶ cellsper well of a 6 well plate. The cells were returned to the incubator andincubated overnight. The media was changed 24 h later. After 72 h thecells were split 1:6 into a 6 well plate. The monolayers were monitoreddaily by light microscopy for evidence of cytopathic effect (CPE). 7-10days post transfection viral plaques were observed and the monolayerharvested by pipetting the media in the wells to lift the cells. Theharvested cells and media were transferred to a 50 mL centrifuge tubefollowed by three rounds of freeze thawing (at −80° C. and 37° C.). Thesubsequent lysate, called the primary virus stock was clarified bycentrifugation at full speed on a bench top centrifuge (4300×g) and aproportion of the lysate 10-50%) used to infect 293A cells in a T25flask. The infected cells were incubated for 48 h before harvestingcells and media at complete CPE. The cells were once again harvested,freeze thawed and clarified before using this secondary viral stock toinfect a T150 flask seeded at 1.5×10⁷ cells per flask. Once complete CPEwas achieved at 72 h the media and cells were harvested and treated aswith earlier viral stocks to generate a tertiary stock.

Production in 293F Cells

ChAdV68 virus production was performed in 293F cells grown in 293FreeStyle™ (ThermoFisher) media in an incubator at 8% CO₂. On the day ofinfection cells were diluted to 10⁶ cells per mL, with 98% viability and400 mL were used per production run in 1L Shake flasks (Corning). 4 mLof the tertiary viral stock with a target MOI of >3.3 was used perinfection. The cells were incubated for 48-72 h until the viability was<70% as measured by Trypan blue. The infected cells were then harvestedby centrifugation, full speed bench top centrifuge and washed in 1×PBS,re-centrifuged and then re-suspended in 20 mL of 10 mM Tris pH7.4. Thecell pellet was lysed by freeze thawing 3× and clarified bycentrifugation at 4,300×g for 5 minutes.

Purification by CsCl Centrifugation

Viral DNA was purified by CsCl centrifugation. Two discontinuousgradient runs were performed. The first to purify virus from cellularcomponents and the second to further refine separation from cellularcomponents and separate defective from infectious particles.

10 mL of 1.2 (26.8 g CsCl dissolved in 92 mL of 10 mM Tris pH 8.0) CsClwas added to polyallomer tubes. Then 8 mL of 1.4 CsCl (53 g CsCldissolved in 87 mL of 10 mM Tris pH 8.0) was carefully added using apipette delivering to the bottom of the tube. The clarified virus wascarefully layered on top of the 1.2 layer. If needed more 10 mM Tris wasadded to balance the tubes. The tubes were then placed in a SW-32Tirotor and centrifuged for 2 h 30 min at 10° C. The tube was then removedto a laminar flow cabinet and the virus band pulled using an 18 gaugeneedle and a 10 mL syringe. Care was taken not to remove contaminatinghost cell DNA and protein. The band was then diluted at least 2× with 10mM Tris pH 8.0 and layered as before on a discontinuous gradient asdescribed above. The run was performed as described before except thatthis time the run was performed overnight. The next day the band waspulled with care to avoid pulling any of the defective particle band.The virus was then dialyzed using a Slide-a-Lyzer™ Cassette (Pierce)against ARM buffer (20 mM Tris pH 8.0, 25 mM NaCl, 2.5% Glycerol). Thiswas performed 3×, 1 h per buffer exchange. The virus was then aliquotedfor storage at −80° C.

Viral Assays

VP concentration was performed by using an OD 260 assay based on theextinction coefficient of 1.1×10¹² viral particles (VP) is equivalent toan Absorbance value of 1 at OD260 nm. Two dilutions (1:5 and 1:10) ofadenovirus were made in a viral lysis buffer (0.1% SDS, 10 mM Tris pH7.4, 1 mM EDTA). OD was measured in duplicate at both dilutions and theVP concentration/mL was measured by multiplying the OD260 value Xdilution factor X 1.1×10¹² VP.

An infectious unit (IU) titer was calculated by a limiting dilutionassay of the viral stock. The virus was initially diluted 100× inDMEM/5% NS/1×PS and then subsequently diluted using 10-fold dilutionsdown to 1×10⁻⁷. 100 μL of these dilutions were then added to 293A cellsthat were seeded at least an hour before at 3e5 cells/well of a 24 wellplate. This was performed in duplicate. Plates were incubated for 48 hin a CO2 (5%) incubator at 37° C. The cells were then washed with 1×PBSand were then fixed with 100% cold methanol (−20° C.). The plates werethen incubated at −20° C. for a minimum of 20 minutes. The wells werewashed with 1×PBS then blocked in 1×PBS/0.1% BSA for 1 h at roomtemperature. A rabbit anti-Ad antibody (Abcam, Cambridge, MA) was addedat 1:8,000 dilution in blocking buffer (0.25 ml per well) and incubatedfor 1 h at room temperature. The wells were washed 4× with 0.5 mL PBSper well. A HRP conjugated Goat anti-Rabbit antibody (Bethyl Labs,Montgomery Texas) diluted 1000× was added per well and incubated for 1 hprior to a final round of washing. 5 PBS washes were performed and theplates were developed using DAB (Diaminobenzidine tetrahydrochloride)substrate in Tris buffered saline (0.67 mg/mL DAB in 50 mM Tris pH 7.5,150 mM NaCl) with 0.01% H₂O₂. Wells were developed for 5 min prior tocounting. Cells were counted under a 10× objective using a dilution thatgave between 4-40 stained cells per field of view. The field of viewthat was used was a 0.32 mm² grid of which there are equivalent to 625per field of view on a 24 well plate. The number of infectiousviruses/mL can be determined by the number of stained cells per gridmultiplied by the number of grids per field of view multiplied by adilution factor 10. Similarly, when working with GFP expressing cellsflorescent can be used rather than capsid staining to determine thenumber of GFP expressing virions per mL.

Immunizations

C57BL/6J female mice and Balb/c female mice were injected with 1×10⁸viral particles (VP) of ChAdV68.5WTnt.MAG25mer in 100 uL volume,bilateral intramuscular injection (50 uL per leg).

Splenocyte Dissociation

Spleen and lymph nodes for each mouse were pooled in 3 mL of completeRPMI (RPMI, 10% FBS, penicillin/streptomycin). Mechanical dissociationwas performed using the gentleMACS Dissociator (Miltenyi Biotec),following manufacturer's protocol. Dissociated cells were filteredthrough a 40 micron filter and red blood cells were lysed with ACK lysisbuffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA). Cells were filteredagain through a 30 micron filter and then resuspended in complete RPMI.Cells were counted on the Attune NxT flow cytometer (Thermo Fisher)using propidium iodide staining to exclude dead and apoptotic cells.Cell were then adjusted to the appropriate concentration of live cellsfor subsequent analysis.

Ex Vivo Enzyme-Linked Immunospot (ELISPOT) Analysis

ELISPOT analysis was performed according to ELISPOT harmonizationguidelines {DOI: 10.1038/nprot.2015.068} with the mouse IFNg ELISpotPLUSkit (MABTECH). 5×10⁴ splenocytes were incubated with 10 uM of theindicated peptides for 16 hours in 96-well IFNg antibody coated plates.Spots were developed using alkaline phosphatase. The reaction was timedfor 10 minutes and was terminated by running plate under tap water.Spots were counted using an AID vSpot Reader Spectrum. For ELISPOTanalysis, wells with saturation >50% were recorded as “too numerous tocount”. Samples with deviation of replicate wells >10% were excludedfrom analysis. Spot counts were then corrected for well confluency usingthe formula: spot count+2×(spot count×% confluence/[100%−% confluence]).Negative background was corrected by subtraction of spot counts in thenegative peptide stimulation wells from the antigen stimulated wells.Finally, wells labeled too numerous to count were set to the highestobserved corrected value, rounded up to the nearest hundred.

XV.B.2. Production of ChAdV68 Viral Delivery Particles after DNATransfection

In one example, ChAdV68.4WTnt.GFP (FIG. 7 ) and ChAdV68.5WTnt.GFP (FIG.8 ) DNA was transfected into HEK293A cells and virus replication (viralplaques) was observed 7-10 days after transfection. ChAdV68 viralplaques were visualized using light (FIGS. 7A and 8A) and fluorescentmicroscopy (FIG. 7B-C and FIG. 8B-C). GFP denotes productive ChAdV68viral delivery particle production.

XV.B.3. ChAdV68 Viral Delivery Particles Expansion

In one example, ChAdV68.4WTnt.GFP, ChAdV68.5WTnt.GFP, andChAdV68.5WTnt.MAG25mer viruses were expanded in HEK293F cells and apurified virus stock produced 18 days after transfection (FIG. 9 ).Viral particles were quantified in the purified ChAdV68 virus stocks andcompared to adenovirus type 5 (Ad5) and ChAdVY25 (a closely relatedChAdV; Dicks, 2012, PloS ONE 7, e40385) viral stocks produced using thesame protocol. ChAdV68 viral titers were comparable to Ad5 and ChAdVY25(Table 7).

TABLE 7 Adenoviral vector production in 293F suspension cells ConstructAverage VP/cell +/− SD Ad5-Vectors (Multiple vectors) 2.96e4 +/− 2.26e4Ad5-GFP 3.89e4 chAdY25-GFP 1.75e3 +/− 6.03e1 ChAdV68.4WTnt.GFP 1.2e4 +/−6.5e3 ChAdV68.5WTnt.GFP 1.8e3  ChAdV68.5WTnt.MAG25mer 1.39e3 +/− 1.1e3*SD is only reported where multiple Production runs have been performed

XV.B.4. Evaluation of Immunogenicity in Tumor Models

C68 vector expressing mouse tumor antigens were evaluated in mouseimmunogenicity studies to demonstrate the C68 vector elicits T-cellresponses. T-cell responses to the MHC class I epitope SIINFEKL (SEQ IDNO: 72) were measured in C57BL/6J female mice and the MHC class Iepitope AH1-A5 (Slansky et al., 2000, Immunity 13:529-538) measured inBalb/c mice. As shown in FIG. 15 , strong T-cell responses relative tocontrol were measured after immunization of mice withChAdV68.5WTnt.MAG25mer. Mean cellular immune responses of 8957 or 4019spot forming cells (SFCs) per 10⁶ splenocytes were observed in ELISpotassays when C57BL/6J or Balb/c mice were immunized withChAdV68.5WTnt.MAG25mer, respectively, 10 days after immunization.

Tumor infiltrating lymphocytes were also evaluated in CT26 tumor modelevaluating ChAdV and co-administration of a an anti-CTLA4 antibody. Micewere implanted with CT26 tumors cells and 7 days after implantation,were immunized with ChAdV vaccine and treated with anti-CTLA4 antibody(clone 9D9) or IgG as a control. Tumor infiltrating lymphocytes wereanalyzed 12 days after immunization. Tumors from each mouse weredissociated using the gentleMACS Dissociator (Miltenyi Biotec) and mousetumor dissociation kit (Miltenyi Biotec). Dissociated cells werefiltered through a 30 micron filter and resuspended in complete RPMI.Cells were counted on the Attune NxT flow cytometer (Thermo Fisher)using propidium iodide staining to exclude dead and apoptotic cells.Cell were then adjusted to the appropriate concentration of live cellsfor subsequent analysis. Antigen specific cells were identified byMHC-tetramer complexes and co-stained with anti-CD8 and a viabilitymarker. Tumors were harvested 12 days after prime immunization.

Antigen-specific CD8+ T cells within the tumor comprised a median of3.3%, 2.2%, or 8.1% of the total live cell population in ChAdV,anti-CTLA4, and ChAdV+anti-CTLA4 treated groups, respectively (FIG. 41and Table 40). Treatment with anti-CTLA in combination with active ChAdVimmunization resulted in a statistically significant increase in theantigen-specific CD8+ T cell frequency over both ChAdV alone andanti-CTLA4 alone demonstrating anti-CTLA4, when co-administered with theChAdV68 vaccine, increased the number of infiltrating T cells within atumor.

TABLE 40 Tetramer+ infiltrating CD8 T cell frequencies in CT26 tumorsTreatment Median % tetramer+ ChAdV68.5WTnt.MAG25mer 3.3 (ChAdV)Anti-CTLA4 2.2 ChAdV68.5WTnt.MAG25mer 8.1 (ChAdV) + anti-CTLA4

XVI. Alphavirus Antigen Cassette Delivery Vector

XVI.A. Alphavirus Delivery Vector Evaluation Materials and Methods

In Vitro Transcription to Generate RNA

For in vitro testing: plasmid DNA was linearized by restriction digestwith PmeI, column purified following manufacturer's protocol (GeneJetDNA cleanup kit, Thermo) and used as template. In vitro transcriptionwas performed using the RiboMAX Large Scale RNA production System(Promega) with the m⁷G cap analog (Promega) according to manufacturer'sprotocol. mRNA was purified using the RNeasy kit (Qiagen) according tomanufacturer's protocol.

For in vivo studies: RNA was generated and purified by TriLinkBiotechnologies and capped with Enzymatic Cap1.

Transfection of RNA

HEK293A cells were seeded at 6e4 cells/well for 96 wells and 2e5cells/well for 24 wells, ˜16 hours prior to transfection. Cells weretransfected with mRNA using MessengerMAX lipofectamine (Invitrogen) andfollowing manufacturer's protocol. For 96-wells, 0.15 uL oflipofectamine and 10 ng of mRNA was used per well, and for 24-wells,0.75 uL of lipofectamine and 150 ng of mRNA was used per well. A GFPexpressing mRNA (TriLink Biotechnologies) was used as a transfectioncontrol.

Luciferase Assay

Luciferase reporter assay was performed in white-walled 96-well plateswith each condition in triplicate using the ONE-Glo luciferase assay(Promega) following manufacturer's protocol. Luminescence was measuredusing the SpectraMax.

qRT-PCR

Transfected cells were rinsed and replaced with fresh media 2 hours posttransfection to remove any untransfected mRNA. Cells were then harvestedat various timepoints in RLT plus lysis buffer (Qiagen), homogenizedusing a QiaShredder (Qiagen) and RNA was extracted using the RNeasy kit(Qiagen), all according to manufacturer's protocol. Total RNA wasquantified using a Nanodrop (Thermo Scientific). qRT-PCR was performedusing the Quantitect Probe One-Step RT-PCR kit (Qiagen) on the qTower³(Analytik Jena) according to manufacturer's protocol, using 20 ng oftotal RNA per reaction. Each sample was run in triplicate for eachprobe. Actin or GusB were used as reference genes. Custom primer/probeswere generated by IDT (Table 8).

TABLE 8  qPCR primers/probes SEQ ID Target NO: Luci Primer1GTGGTGTGCAGCGAGAATAG 178 Primer2 CGCTCGTTGTAGATGTCGTTAG 179 Probe/56-FAM/TTGCAGTTC/ZEN/ 180 TTCATGCCCGTGTTG/3IABkFQ/ GusB Primer1GTTTTTGATCCAGACCCAGATG 181 Primer2 GCCCATTATTCAGAGCGAGTA 182 Probe/56-FAM/TGCAGGGTT/ZEN/ 183 TCACCAGGATCCAC/3IABkFQ/ ActB Primer1CCTTGCACATGCCGGAG 184 Primer2 ACAGAGCCTCGCCTTTG 185 Probe/56-FAM/TCATCCATG/ZEN/ 186 GTGAGCTGGCGG/3IABkFQ/ MAG- Primer1CTGAAAGCTCGGTTTGCTAATG 187 25mer Primer2 CCATGCTGGAAGAGACAATCT 188 Set1Probe /56-FAM/CGTTTCTGA/ZEN/ 189 TGGCGCTGACCGATA/3IABkFQ/ MAG- Primer1TATGCCTATCCTGTCTCCTCTG 190 25mer Primer2 GCTAATGCAGCTAAGTCCTCTC 191 Set2Probe /56-FAM/TGTTTACCC/ZEN/ 192 TGACCGTGCCTTCTG/3IABkFQ/

B16-OVA Tumor Model

C57BL/6J mice were injected in the lower left abdominal flank with 10⁵B16-OVA cells/animal. Tumors were allowed to grow for 3 days prior toimmunization.

CT26 Tumor Model

Balb/c mice were injected in the lower left abdominal flank with 10⁶CT26 cells/animal. Tumors were allowed to grow for 7 days prior toimmunization.

Immunizations

For srRNA vaccine, mice were injected with 10 ug of RNA in 100 uLvolume, bilateral intramuscular injection (50 uL per leg). For Ad5vaccine, mice were injected with 5×10¹⁰ viral particles (VP) in 100 uLvolume, bilateral intramuscular injection (50 uL per leg). Animals wereinjected with anti-CTLA-4 (clone 9D9, BioXcell), anti-PD-1 (cloneRMP1-14, BioXcell) or anti-IgG (clone MPC-11, BioXcell), 250 ug dose, 2times per week, via intraperitoneal injection.

In Vivo Bioluminescent Imaging

At each timepoint mice were injected with 150 mg/kg luciferin substratevia intraperitoneal injection and bioluminescence was measured using theIVIS In vivo imaging system (PerkinElmer) 10-15 minutes after injection.

Splenocyte Dissociation

Spleen and lymph nodes for each mouse were pooled in 3 mL of completeRPMI (RPMI, 10% FBS, penicillin/streptomycin). Mechanical dissociationwas performed using the gentleMACS Dissociator (Miltenyi Biotec),following manufacturer's protocol. Dissociated cells were filteredthrough a 40 micron filter and red blood cells were lysed with ACK lysisbuffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA). Cells were filteredagain through a 30 micron filter and then resuspended in complete RPMI.Cells were counted on the Attune NxT flow cytometer (Thermo Fisher)using propidium iodide staining to exclude dead and apoptotic cells.Cell were then adjusted to the appropriate concentration of live cellsfor subsequent analysis.

Ex Vivo Enzyme-Linked Immunospot (ELISPOT) Analysis

ELISPOT analysis was performed according to ELISPOT harmonizationguidelines {DOI: 10.1038/nprot.2015.068} with the mouse IFNg ELISpotPLUSkit (MABTECH). 5×10⁴ splenocytes were incubated with 10 uM of theindicated peptides for 16 hours in 96-well IFNg antibody coated plates.Spots were developed using alkaline phosphatase. The reaction was timedfor 10 minutes and was terminated by running plate under tap water.Spots were counted using an AID vSpot Reader Spectrum. For ELISPOTanalysis, wells with saturation >50% were recorded as “too numerous tocount”. Samples with deviation of replicate wells >10% were excludedfrom analysis. Spot counts were then corrected for well confluency usingthe formula: spot count+2×(spot count×% confluence/[100%−% confluence]).Negative background was corrected by subtraction of spot counts in thenegative peptide stimulation wells from the antigen stimulated wells.Finally, wells labeled too numerous to count were set to the highestobserved corrected value, rounded up to the nearest hundred.

XVI.B. Alphavirus Vector

XVI.B.1. Alphavirus Vector In Vitro Evaluation

In one implementation of the present invention, a RNA alphavirusbackbone for the antigen expression system was generated from aVenezuelan Equine Encephalitis (VEE) (Kinney, 1986, Virology 152:400-413) based self-replicating RNA (srRNA) vector. In one example, thesequences encoding the structural proteins of VEE located 3′ of the 26Ssubgenomic promoter were deleted (VEE sequences 7544 to 11,175 deleted;numbering based on Kinney et al 1986; SEQ ID NO:6) and replaced byantigen sequences (SEQ ID NO:14 and SEQ ID NO:4) or a luciferasereporter (e.g., VEE-Luciferase, SEQ ID NO:15) (FIG. 10 ). RNA wastranscribed from the srRNA DNA vector in vitro, transfected into HEK293Acells and luciferase reporter expression was measured. In addition, an(non-replicating) mRNA encoding luciferase was transfected forcomparison. An ˜30,000-fold increase in srRNA reporter signal wasobserved for VEE-Luciferase srRNA when comparing the 23 hour measurementvs the 2 hour measurement (Table 9). In contrast, the mRNA reporterexhibited a less than 10-fold increase in signal over the same timeperiod (Table 9).

TABLE 9 Expression of luciferase from VEE self-replicating vectorincreases over time. HEK293A cells transfected with 10 ng ofVEE-Luciferase srRNA or 10 ng of non-replicating luciferase mRNA(TriLink L-6307) per well in 96 wells. Luminescence was measured atvarious times post transfection. Luciferase expression is reported asrelative luminescence units (RLU). Each data point is the mean +/− SD of3 transfected wells. Timepoint Standard Dev Construct (hr) Mean RLUtriplicate wells) mRNA 2 878.6666667 120.7904522 mRNA 5 1847.333333978.515372 mRNA 9 4847 868.3271273 mRNA 23 8639.333333 751.6816702 SRRNA2 27 15 SRRNA 5 4884.333333 2955.158935 SRRNA 9 182065.5 16030.81784SRRNA 23 783658.3333 68985.05538

In another example, replication of the srRNA was confirmed directly bymeasuring RNA levels after transfection of either the luciferaseencoding srRNA (VEE-Luciferase) or an srRNA encoding a multi-epitopecassette (VEE-MAG25mer) using quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). An ˜150-fold increase in RNA wasobserved for the VEE-luciferase srRNA (Table 10), while a 30-50-foldincrease in RNA was observed for the VEE-MAG25mer srRNA (Table 11).These data confirm that the VEE srRNA vectors replicate when transfectedinto cells.

TABLE 10 Direct measurement of RNA replication in VEE-Luciferase srRNAtransfected cells. HEK293A cells transfected with VEE-Luciferase srRNA(150 ng per well, 24-well) and RNA levels quantified by qRT-PCR atvarious times after transfection. Each measurement was normalized basedon the Actin reference gene and fold-change relative to the 2 hourtimepoint is presented. Timepoint Luciferase Actin Ref Relative Fold(hr) Ct Ct dCt dCt ddCt change 2 20.51 18.14 2.38 2.38 0.00 1.00 4 20.0918.39 1.70 2.38 −0.67 1.59 6 15.50 18.19 −2.69 2.38 −5.07 33.51 8 13.5118.36 −4.85 2.38 −7.22 149.43

TABLE 11 Direct measurement of RNA replication in VEE-MAG25mer srRNAtransfected cells. HEK293 cells transfected with VEE-MAG25mer srRNA (150ng per well, 24-well) and RNA levels quantified by qRT-PCR at varioustimes after transfection. Each measurement was normalized based on theGusB reference gene and fold- change relative to the 2 hour timepoint ispresented. Different lines on the graph represent 2 different qPCRprimer/probe sets, both of which detect the enitone cassette region ofthe srRNA. Primer/ Timepoint GusB Ref Relative probe (hr) Ct Ct dCt dCtddCt Fold-Change Set1 2 18.96 22.41 −3.45 −3.45 0.00 1.00 Set1 4 17.4622.27 −4.81 −3.45 −1.37 2.58 Set1 6 14.87 22.04 −7.17 −3.45 −3.72 13.21Set1 8 14.16 22.19 −8.02 −3.45 −4.58 23.86 Set1 24 13.16 22.01 −8.86−3.45 −5.41 42.52 Set1 36 13.53 22.63 −9.10 −3.45 −5.66 50.45 Set2 217.75 22.41 −4.66 −4.66 0.00 1.00 Set2 4 16.66 22.27 −5.61 −4.66 −0.941.92 Set2 6 14.22 22.04 −7.82 −4.66 −3.15 8.90 Set2 8 13.18 22.19 −9.01−4.66 −4.35 20.35 Set2 24 12.22 22.01 −9.80 −4.66 −5.13 35.10 Set2 3613.08 22.63 −9.55 −4.66 −4.89 29.58

XVI.B.2. Alphavirus Vector in vivo Evaluation

In another example, VEE-Luciferase reporter expression was evaluated invivo. Mice were injected with 10 ug of VEE-Luciferase srRNA encapsulatedin lipid nanoparticle (MC3) and imaged at 24 and 48 hours, and 7 and 14days post injection to determine bioluminescent signal. Luciferasesignal was detected at 24 hours post injection and increased over timeand appeared to peak at 7 days after srRNA injection (FIG. 11 ).

XVI.B.3. Alphavirus Vector Tumor Model Evaluation

In one implementation, to determine if the VEE srRNA vector directsantigen-specific immune responses in vivo, a VEE srRNA vector wasgenerated (VEE-UbAAY, SEQ ID NO:14) that expresses 2 different MHC classI mouse tumor epitopes, SIINFEKL (SEQ ID NO: 72) and AH1-A5 (Slansky etal., 2000, Immunity 13:529-538). The SFL (SIINFEKL (SEQ ID NO: 72))epitope is expressed by the B16-OVA melanoma cell line, and the AH1-A5(SPSYAYHQF (SEQ ID NO: 73); Slansky et al., 2000, Immunity) epitopeinduces T cells targeting a related epitope (AH1/SPSYVYHQF (SEQ ID NO:193); Huang et al., 1996, Proc Natl Acad Sci USA 93:9730-9735) that isexpressed by the CT26 colon carcinoma cell line. In one example, for invivo studies, VEE-UbAAY srRNA was generated by in vitro transcriptionusing T7 polymerase (TriLink Biotechnologies) and encapsulated in alipid nanoparticle (MC3).

A strong antigen-specific T-cell response targeting SFL, relative tocontrol, was observed two weeks after immunization of B16-OVA tumorbearing mice with MC3 formulated VEE-UbAAY srRNA. In one example, amedian of 3835 spot forming cells (SFC) per 10⁶ splenocytes was measuredafter stimulation with the SFL peptide in ELISpot assays (FIG. 12A,Table 12) and 1.8% (median) of CD8 T-cells were SFL antigen-specific asmeasured by pentamer staining (FIG. 12B, Table 12). In another example,co-administration of an anti-CTLA-4 monoclonal antibody (mAb) with theVEE srRNA vaccine resulted in a moderate increase in overall T-cellresponses with a median of 4794.5 SFCs per 10⁶ splenocytes measured inthe ELISpot assay (FIG. 12A, Table 12).

TABLE 12 Results of ELISPOT and MHCI-pentamer staining assays 14 dayspost VEE srRNA immunization in B16-OVA tumor bearing C57BL/6J mice.Pentamer Pentamer SFC/1e6 positive SFC/1e6 positive Group Mousesplenocytes (% of CD8) Group Mouse splenocytes (% of CD8) Control 1 470.22 Vax 1 6774 4.92 2 80 0.32 2 2323 1.34 3 0 0.27 3 2997 1.52 4 0 0.294 4492 1.86 5 0 0.27 5 4970 3.7 6 0 0.25 6 4.13 7 0 0.23 7 3835 1.66 887 0.25 8 3119 1.64 aCTLA4 1 0 0.24 Vax + 1 6232 2.16 2 0 0.26 aCTLA4 24242 0.82 3 0 0.39 3 5347 1.57 4 0 0.28 4 6568 2.33 5 0 0.28 5 6269 1.556 0 0.28 6 4056 1.74 7 0 0.31 7 4163 1.14 8 6 0.26 8 3667 1.01 * Notethat results from mouse #6 in the Vax group were excluded from analysisdue to high variability between triplicate wells.

In another implementation, to mirror a clinical approach, a heterologousprime/boost in the B16-OVA and CT26 mouse tumor models was performed,where tumor bearing mice were immunized first with adenoviral vectorexpressing the same antigen cassette (Ad5-UbAAY), followed by a boostimmunization with the VEE-UbAAY srRNA vaccine 14 days after theAd5-UbAAY prime. In one example, an antigen-specific immune response wasinduced by the Ad5-UbAAY vaccine resulting in 7330 (median) SFCs per 10⁶splenocytes measured in the ELISpot assay (FIG. 13A, Table 13) and 2.9%(median) of CD8 T-cells targeting the SFL antigen as measured bypentamer staining (FIG. 13C, Table 13). In another example, the T-cellresponse was maintained 2 weeks after the VEE-UbAAY srRNA boost in theB16-OVA model with 3960 (median) SFL-specific SFCs per 10⁶ splenocytesmeasured in the ELISpot assay (FIG. 13B, Table 13) and 3.1% (median) ofCD8 T-cells targeting the SFL antigen as measured by pentamer staining(FIG. 13D, Table 13).

TABLE 13 Immune monitoring of B16-OVA mice following heterologousprime/boost with Ad5 vaccine prime and srRNA boost. Pentamer PentamerSFC/1e6 positive SFC/1e6 positive Group Mouse splenocytes (% of CD8)Group Mouse splenocytes (% of CD8) Day 14 Control 1 0 0.10 Vax 1 85141.87 2 0 0.09 2 7779 1.91 3 0 0.11 3 6177 3.17 4 46 0.18 4 7945 3.41 5 00.11 5 8821 4.51 6 16 0.11 6 6881 2.48 7 0 0.24 7 5365 2.57 8 37 0.10 86705 3.98 aCTLA4 1 0 0.08 Vax + 1 9416 2.35 2 29 0.10 aCTLA4 2 7918 3.333 0 0.09 3 10153 4.50 4 29 0.09 4 7212 2.98 5 0 0.10 5 11203 4.38 6 490.10 6 9784 2.27 7 0 0.10 8 7267 2.87 8 31 0.14 Day 28 Control 2 0 0.17Vax 1 5033 2.61 4 0 0.15 2 3958 3.08 6 20 0.17 4 3960 3.58 aCTLA4 1 70.23 Vax + 4 3460 2.44 2 0 0.18 aCTLA4 5 5670 3.46 3 0 0.14

In another implementation, similar results were observed after anAd5-UbAAY prime and VEE-UbAAY srRNA boost in the CT26 mouse model. Inone example, an AH1 antigen-specific response was observed after theAd5-UbAAY prime (day 14) with a mean of 5187 SFCs per 10⁶ splenocytesmeasured in the ELISpot assay (FIG. 14A, Table 14) and 3799 SFCs per 10⁶splenocytes measured in the ELISpot assay after the VEE-UbAAY srRNAboost (day 28) (FIG. 14B, Table 14).

TABLE 14 Immune monitoring after heterologous prime/boost in CT26 tumormouse model. Day 12 Day 21 SFC/1e6 SFC/1e6 Group Mouse splenocytes GroupMouse splenocytes Control 1 1799 Control 9 167 2 1442 10 115 3 1235 11347 aPD1 1 737 aPD1 8 511 2 5230 11 758 3 332 Vax 9 3133 Vax 1 6287 102036 2 4086 11 6227 Vax + 1 5363 Vax+ 8 3844 aPD1 2 6500 aPD1 9 2071 114888

XVII. ChAdV/srRNA Combination Tumor Model Evaluation

Various dosing protocols using ChAdV68 and self-replicating RNA (srRNA)were evaluated in murine CT26 tumor models.

XVII.a ChAdV/srRNA Combination Tumor Model Evaluation Methods andMaterials

Tumor Injection

Balb/c mice were injected with the CT26 tumor cell line. 7 days aftertumor cell injection, mice were randomized to the different study arms(28-40 mice per group) and treatment initiated. Balb/c mice wereinjected in the lower left abdominal flank with 10⁶ CT26 cells/animal.Tumors were allowed to grow for 7 days prior to immunization. The studyarms are described in detail in Table 15.

TABLE 15 ChAdV/srRNA Combination Tumor Model Evaluation Study Arms GroupN Treatment Dose Volume Schedule Route 1 40 ChAdV68 control 1e11 vp 2 ×50 uL day 0 IM srRNA control 10 ug 50 uL day 14, 28, 42 IM Anti-PD1 250ug 100 uL 2 ×/week (start day 0) IP 2 40 ChAdV68 control 1e11 vp 2 × 50uL day 0 IM srRNA control 10 ug 50 uL day 14, 28, 42 IM Anti-IgG 250 ug100 uL 2 ×/week (start day 0) IP 3 28 ChAdV68 vaccine 1e11 vp 2 × 50 uLday 0 IM srRNA vaccine 10 ug 50 uL day 14, 28, 42 IM Anti-PD1 250 ug 100uL 2 ×/week (start day 0) IP 4 28 ChAdV68 vaccine 1e11 vp 2 × 50 uL day0 IM srRNA vaccine 10 ug 50 uL day 14, 28, 42 IM Anti-IgG 250 ug 100 uL2 ×/week (start day 0) IP 5 28 srRNA vaccine 10 ug 50 uL day 0, 28, 42IM ChAdV68 vaccine 1e11 vp 2 × 50 uL day 14 IM Anti-PD1 250 ug 100 uL 2×/week (start day 0) IP 6 28 srRNA vaccine 10 ug 50 uL day 0, 28, 42 IMChAdV68 vaccine 1e11 vp 2 × 50 uL day 14 IM Anti-IgG 250 ug 100 uL 2×/week (start day 0) IP 7 40 srRNA vaccine 10 ug 50 uL day 0, 14, 28, 42IM Anti-PD1 250 ug 100 uL 2 ×/week (start day 0) IP 8 40 srRNA vaccine10 ug 50 uL day 0, 14, 28, 42 IM Anti-IgG 250 ug 100 uL 2 ×/week (startday 0) IP

Immunizations

For srRNA vaccine, mice were injected with 10 ug of VEE-MAG25mer srRNAin 100 uL volume, bilateral intramuscular injection (50 uL per leg). ForC68 vaccine, mice were injected with 1×10¹¹ viral particles (VP) ofChAdV68.5WTnt.MAG25mer in 100 uL volume, bilateral intramuscularinjection (50 uL per leg). Animals were injected with anti-PD-1 (cloneRMP1-14, BioXcell) or anti-IgG (clone MPC-11, BioXcell), 250 ug dose, 2times per week, via intraperitoneal injection.

Splenocyte Dissociation

Spleen and lymph nodes for each mouse were pooled in 3 mL of completeRPMI (RPMI, 10% FBS, penicillin/streptomycin). Mechanical dissociationwas performed using the gentleMACS Dissociator (Miltenyi Biotec),following manufacturer's protocol. Dissociated cells were filteredthrough a 40 micron filter and red blood cells were lysed with ACK lysisbuffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA). Cells were filteredagain through a 30 micron filter and then resuspended in complete RPMI.Cells were counted on the Attune NxT flow cytometer (Thermo Fisher)using propidium iodide staining to exclude dead and apoptotic cells.Cell were then adjusted to the appropriate concentration of live cellsfor subsequent analysis.

Ex Vivo Enzyme-Linked Immunospot (ELISPOT) Analysis

ELISPOT analysis was performed according to ELISPOT harmonizationguidelines {DOI: 10.1038/nprot.2015.068} with the mouse IFNg ELISpotPLUSkit (MABTECH). 5×10⁴ splenocytes were incubated with 10 uM of theindicated peptides for 16 hours in 96-well IFNg antibody coated plates.Spots were developed using alkaline phosphatase. The reaction was timedfor 10 minutes and was terminated by running plate under tap water.Spots were counted using an AID vSpot Reader Spectrum. For ELISPOTanalysis, wells with saturation >50% were recorded as “too numerous tocount”. Samples with deviation of replicate wells >10% were excludedfrom analysis. Spot counts were then corrected for well confluency usingthe formula: spot count+2×(spot count×% confluence/[100%−% confluence]).Negative background was corrected by subtraction of spot counts in thenegative peptide stimulation wells from the antigen stimulated wells.Finally, wells labeled too numerous to count were set to the highestobserved corrected value, rounded up to the nearest hundred.

XVI.B ChAdV/srRNA Combination Evaluation in a CT26 Tumor Model

The immunogenicity and efficacy of theChAdV68.5WTnt.MAG25mer/VEE-MAG25mer srRNA heterologous prime/boost orVEE-MAG25mer srRNA homologous prime/boost vaccines were evaluated in theCT26 mouse tumor model. Balb/c mice were injected with the CT26 tumorcell line. 7 days after tumor cell injection, mice were randomized tothe different study arms and treatment initiated. The study arms aredescribed in detail in Table 15 and more generally in Table 16.

TABLE 16 Prime/Boost Study Arms Group Prime Boost 1 Control Control 2Control + anti-PD-1 Control + anti-PD-1 3 ChAdV68.5WTnt.MAG25merVEE-MAG25mer srRNA 4 ChAdV68.5WTnt.MAG25mer + VEE-MAG25mer srRNA +anti-PD-1 anti-PD-1 5 VEE-MAG25mer srRNA ChAdV68.5WTnt.MAG25mer 6VEE-MAG25mer srRNA + ChAdV68.5WTnt.MAG25mer + anti-PD-1 anti-PD-1 7VEE-MAG25mer srRNA VEE-MAG25mer srRNA 8 VEE-MAG25mer srRNA +VEE-MAG25mer srRNA + anti-PD-1 anti-PD-1

Spleens were harvested 14 days after the prime vaccination for immunemonitoring. Tumor and body weight measurements were taken twice a weekand survival was monitored. Strong immune responses relative to controlwere observed in all active vaccine groups.

Median cellular immune responses of 10,630, 12,976, 3319, or 3745 spotforming cells (SFCs) per 10⁶ splenocytes were observed in ELISpot assaysin mice immunized with ChAdV68.5WTnt.MAG25mer (ChAdV/group 3),ChAdV68.5WTnt.MAG25mer+anti-PD-1 (ChAdV+PD-1/group 4), VEE-MAG25mersrRNA (srRNA/median for groups 5 & 7 combined), or VEE-MAG25mersrRNA+anti-PD-1 (srRNA+PD-1/median for groups 6 & 8 combined),respectively, 14 days after the first immunization (FIG. 16 and Table17). In contrast, the vaccine control (group 1) or vaccine control withanti-PD-1 (group 2) exhibited median cellular immune responses of 296 or285 SFC per 10⁶ splenocytes, respectively.

TABLE 17 Cellular immune responses in a CT26 tumor model TreatmentMedian SFC/10⁶ Splenocytes Control 296 PD1 285 ChAdV68.5WTnt.MAG25mer10630 (ChAdV) ChAdV68.5WTnt.MAG25mer + 12976 PD1 (ChAdV + PD-1)VEE-MAG25mer srRNA 3319 (srRNA) VEE-MAG25mer srRNA + 3745 PD-1 (srRNA +PD1)

Consistent with the ELISpot data, 5.6, 7.8, 1.8 or 1.9% of CD8 T cells(median) exhibited antigen-specific responses in intracellular cytokinestaining (ICS) analyses for mice immunized with ChAdV68.5WTnt.MAG25mer(ChAdV/group 3), ChAdV68.5WTnt.MAG25mer+anti-PD-1 (ChAdV+PD-1/group 4),VEE-MAG25mer srRNA (srRNA/median for groups 5 & 7 combined), orVEE-MAG25mer srRNA+anti-PD-1 (srRNA+PD-1/median for groups 6 & 8combined), respectively, 14 days after the first immunization (FIG. 17and Table 18). Mice immunized with the vaccine control or vaccinecontrol combined with anti-PD-1 showed antigen-specific CD8 responses of0.2 and 0.1%, respectively.

TABLE 18 CD8 T-Cell responses in a CT26 tumor model Median % CD8 IFN-Treatment gamma Positive Control 0.21 PD1 0.1 ChAdV68.5WTnt.MAG25mer 5.6(ChAdV) ChAdV68.5WTnt.MAG25mer + 7.8 PD1 (ChAdV + PD-1) VEE-MAG25mersrRNA 1.8 (srRNA) VEE-MAG25mer srRNA + 1.9 PD-1 (srRNA + PD1)

Tumor growth was measured in the CT26 colon tumor model for all groups,and tumor growth up to 21 days after treatment initiation (28 days afterinjection of CT-26 tumor cells) is presented. Mice were sacrificed 21days after treatment initiation based on large tumor sizes (>2500 mm³);therefore, only the first 21 days are presented to avoid analyticalbias. Mean tumor volumes at 21 days were 1129, 848, 2142, 1418, 2198 and1606 mm³ for ChAdV68.5WTnt.MAG25mer prime/VEE-MAG25mer srRNA boost(group 3), ChAdV68.5WTnt.MAG25mer prime/VEE-MAG25mer srRNAboost+anti-PD-1 (group 4), VEE-MAG25mer srRNAprime/ChAdV68.5WTnt.MAG25mer boost (group 5), VEE-MAG25mer srRNAprime/ChAdV68.5WTnt.MAG25mer boost+anti-PD-1 (group 6), VEE-MAG25mersrRNA prime/VEE-MAG25mer srRNA boost (group 7) and VEE-MAG25mer srRNAprime/VEE-MAG25mer srRNA boost+anti-PD-1 (group 8), respectively (FIG.18 and Table 19). The mean tumor volumes in the vaccine control orvaccine control combined with anti-PD-1 were 2361 or 2067 mm³,respectively. Based on these data, vaccine treatment withChAdV68.5WTnt.MAG25mer/VEE-MAG25mer srRNA (group 3),ChAdV68.5WTnt.MAG25mer/VEE-MAG25mer srRNA+anti-PD-1 (group 4),VEE-MAG25mer srRNA/ChAdV68.5WTnt.MAG25mer+anti-PD-1 (group 6) andVEE-MAG25mer srRNA/VEE-MAG25mer srRNA+anti-PD-1 (group 8) resulted in areduction of tumor growth at 21 days that was significantly differentfrom the control (group 1).

TABLE 19 Tumor size at day 21 measured in the CT26 model Treatment TumorSize (mm³) SEM Control 2361 235 PD1 2067 137 chAdV/srRNA 1129 181chAdV/srRNA + PD1 848 182 srRNA/chAdV 2142 233 srRNA/chAdV + PD1 1418220 srRNA 2198 134 srRNA + PD1 1606 210

Survival was monitored for 35 days after treatment initiation in theCT-26 tumor model (42 days after injection of CT-26 tumor cells).Improved survival was observed after vaccination of mice with 4 of thecombinations tested. After vaccination, 64%, 46%, 41% and 36% of micesurvived with ChAdV68.5WTnt.MAG25mer prime/VEE-MAG25mer srRNA boost incombination with anti-PD-1 (group 4; P<0.0001 relative to control group1), VEE-MAG25mer srRNA prime/VEE-MAG25mer srRNA boost in combinationwith anti-PD-1 (group 8; P=0.0006 relative to control group 1),ChAdV68.5WTnt.MAG25mer prime/VEE-MAG25mer srRNA boost (group 3; P=0.0003relative to control group 1) and VEE-MAG25mer srRNAprime/ChAdV68.5WTnt.MAG25mer boost in combination with anti-PD-1 (group6; P=0.0016 relative to control group 1), respectively (FIG. 19 andTable 20). Survival was not significantly different from the controlgroup 1 (≤14%) for the remaining treatment groups [VEE-MAG25mer srRNAprime/ChAdV68.5WTnt.MAG25mer boost (group 5), VEE-MAG25mer srRNAprime/VEE-MAG25mer srRNA boost (group 7) and anti-PD-1 alone (group 2)].

TABLE 20 Survival in the CT26 model chAdV/ srRNA/ chAdV/ srRNA + srRNA/chAdV + srRNA + Timepoint Control PD1 srRNA PD1 chAdV PD1 srRNA PD1  0100 100 100 100.00 100.00 100 100 100 21 96 100 100 100 100 95 100 10024 54 64 91 100 68 82 68 71 28 21 32 68 86 45 68 21 64 31 7 14 41 64 1436 11 46 35 7 14 41 64 14 36 11 46

In conclusion, ChAdV68.5WTnt.MAG25mer and VEE-MAG25mer srRNA elicitedstrong T-cell responses to mouse tumor antigens encoded by the vaccines,relative to control. Administration of a ChAdV68.5WTnt.MAG25mer primeand VEE-MAG25mer srRNA boost with or without co-administration ofanti-PD-1, VEE-MAG25mer srRNA prime and ChAdV68.5WTnt.MAG25mer boost incombination with anti-PD-1 or administration of VEE-MAG25mer srRNA as ahomologous prime boost immunization in combination with anti-PD-1 totumor bearing mice resulted in improved survival.

XVIII. Non-Human Primate Studies

Various dosing protocols using ChAdV68 and self-replicating RNA (srRNA)were evaluated in non-human primates (NHP).

Materials and Methods

A priming vaccine was injected intramuscularly (IM) in each NHP toinitiate the study (vaccine prime). One or more boosting vaccines(vaccine boost) were also injected intramuscularly in each NHP.Bilateral injections per dose were administered according to groupsoutlined in tables and summarized below.

Immunizations

Mamu-A*01 Indian rhesus macaques were immunized bilaterally with 1×10¹²viral particles (5×10¹¹ viral particles per injection) ofChAdV68.5WTnt.MAG25mer, 30 ug of VEE-MAG25MER srRNA, 100 ug ofVEE-MAG25mer srRNA or 300 ug of VEE-MAG25mer srRNA formulated in LNP-1or LNP-2. Vaccine boosts of 30 ug, 100 ug or 300 ug VEE-MAG25mer srRNAwere administered intramuscularly at the indicated time after primevaccination.

Immune Monitoring

PBMCs were isolated at indicated times after prime vaccination usingLymphocyte Separation Medium (LSM, MP Biomedicals) and LeucoSepseparation tubes (Greiner Bio-One) and resuspended in RPMI containing10% FBS and penicillin/streptomycin. Cells were counted on the AttuneNxT flow cytometer (Thermo Fisher) using propidium iodide staining toexclude dead and apoptotic cells. Cell were then adjusted to theappropriate concentration of live cells for subsequent analysis. Foreach monkey in the studies, T cell responses were measured using ELISpotor flow cytometry methods. T cell responses to 6 different rhesusmacaque Mamu-A*01 class I epitopes encoded in the vaccines weremonitored from PBMCs by measuring induction of cytokines, such asIFN-gamma, using ex vivo enzyme-linked immunospot (ELISpot) analysis.ELISpot analysis was performed according to ELISPOT harmonizationguidelines {DOI: 10.1038/nprot.2015.068} with the monkey IFNgELISpotPLUS kit (MABTECH). 200,000 PBMCs were incubated with 10 uM ofthe indicated peptides for 16 hours in 96-well IFNg antibody coatedplates. Spots were developed using alkaline phosphatase. The reactionwas timed for 10 minutes and was terminated by running plate under tapwater. Spots were counted using an AID vSpot Reader Spectrum. ForELISPOT analysis, wells with saturation >50% were recorded as “toonumerous to count”. Samples with deviation of replicate wells >10% wereexcluded from analysis. Spot counts were then corrected for wellconfluency using the formula: spot count+2×(spot count×%confluence/[100%−% confluence]). Negative background was corrected bysubtraction of spot counts in the negative peptide stimulation wellsfrom the antigen stimulated wells. Finally, wells labeled too numerousto count were set to the highest observed corrected value, rounded up tothe nearest hundred.

Specific CD4 and CD8 T cell responses to 6 different rhesus macaqueMamu-A*01 class I epitopes encoded in the vaccines were monitored fromPBMCs by measuring induction of intracellular cytokines, such asIFN-gamma, using flow cytometry. The results from both methods indicatethat cytokines were induced in an antigen-specific manner to epitopes.

Immunogenicity in Rhesus Macaques

This study was designed to (a) evaluate the immunogenicity andpreliminary safety of VEE-MAG25mer srRNA 30 μg and 100 μg doses as ahomologous prime/boost or heterologous prime/boost in combination withChAdV68.5WTnt.MAG25mer; (b) compare the immune responses of VEE-MAG25mersrRNA in lipid nanoparticles using LNP1 versus LNP2; (c) evaluate thekinetics of T-cell responses to VEE-MAG25mer srRNA andChAdV68.5WTnt.MAG25mer immunizations.

The study arm was conducted in Mamu-A*01 Indian rhesus macaques todemonstrate immunogenicity. Select antigens used in this study are onlyrecognized in Rhesus macaques, specifically those with a Mamu-A*01 MHCclass I haplotype. Mamu-A*01 Indian rhesus macaques were randomized tothe different study arms (6 macaques per group) and administered an IMinjection bilaterally with either ChAdV68.5WTnt.MAG25mer or VEE-MAG25mersrRNA vector encoding model antigens that includes multiple Mamu-A*01restricted epitopes. The study arms were as described below.

TABLE 21 Non-GLP immunogenicity study in Indian Rhesus Macaques GroupPrime Boost 1 Boost 2 1 VEE-MAG25mer VEE-MAG25mer VEE-MAG25mer srRNA-srRNA- srRNA- LNP1(30 μg) LNP1 (30 μg) LNP1 (30 μg) 2 VEE-MAG25merVEE-MAG25mer VEE-MAG25mer srRNA- srRNA- srRNA- LNP1 (100 μg) LNP1 (100μg) LNP1 (100 μg) 3 VEE-MAG25mer VEE-MAG25mer VEE-MAG25mer srRNA- srRNA-srRNA- LNP2 (100 μg) LNP2 (100 μg) LNP2 (100 μg) 4 ChAdV68.5WTnt.VEE-MAG25mer VEE-MAG25mer MAG25mer srRNA- srRNA- LNP1 (100 μg) LNP1 (100μg)

PBMCs were collected prior to immunization and on weeks 1, 2, 3, 4, 5,6, 8, 9, and 10 after the initial immunization for immune monitoring.

Results

Antigen-specific cellular immune responses in peripheral bloodmononuclear cells (PBMCs) were measured to six different Mamu-A*01restricted epitopes prior to immunization and 1, 2, 3, 4, 5, 6, 8, 9,and 10 weeks after the initial immunization. Animals received a boostimmunization with VEE-MAG25mer srRNA on weeks 4 and 8 with either 30 μgor 100 μg doses, and either formulated with LNP1 or LNP2, as describedin Table 21. Combined immune responses to all six epitopes were plottedfor each immune monitoring timepoint (FIG. 20A-D and Tables 22-25).

Combined antigen-specific immune responses were observed at allmeasurements with 170, 14, 15, 11, 7, 8, 14, 17, 12 SFCs per 10⁶ PBMCs(six epitopes combined) 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after aninitial VEE-MAG25mer srRNA-LNP1(30 μg) prime immunization, respectively(FIG. 20A). Combined antigen-specific immune responses were observed atall measurements with 108, −3, 14, 1, 37, 4, 105, 17, 25 SFCs per 10⁶PBMCs (six epitopes combined) 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks afteran initial VEE-MAG25mer srRNA-LNP1(100 μg) prime immunization,respectively (FIG. 20B). Combined antigen-specific immune responses wereobserved at all measurements with −17, 38, 14, −2, 87, 21, 104, 129, 89SFCs per 10⁶ PBMCs (six epitopes combined) 1, 2, 3, 4, 5, 6, 8, 9, or 10weeks after an initial VEE-MAG25mer srRNA-LNP2(100 μg) primeimmunization, respectively (FIG. 20C). Negative values are a result ofnormalization to pre-bleed values for each epitope/animal.

Combined antigen-specific immune responses were observed at allmeasurements with 1218, 1784, 1866, 973, 1813, 747, 797, 1249, and 547SFCs per 10⁶ PBMCs (six epitopes combined) 1, 2, 3, 4, 5, 6, 8, 9, or 10weeks after an initial ChAdV68.5WTnt.MAG25mer prime immunization,respectively (FIG. 20D). The immune response showed the expected profilewith peak immune responses measured ˜2-3 weeks after the primeimmunization followed by a contraction in the immune response after 4weeks. Combined antigen-specific cellular immune responses of 1813 SFCsper 10⁶ PBMCs (six epitopes combined) were measured 5 weeks after theinitial immunization with ChAdV68.5WTnt.MAG25mer (i.e., 1 week after thefirst boost with VEE-MAG25mer srRNA). The immune response measured 1week after the first boost with VEE-MAG25mer srRNA (week 5) wascomparable to the peak immune response measured for theChAdV68.5WTnt.MAG25mer prime immunization (week 3) (FIG. 20D). Combinedantigen-specific cellular immune responses of 1249 SFCs per 10⁶ PBMCs(six epitopes combined) was measured 9 weeks after the initialimmunization with ChAdV68.5WTnt.MAG25mer, respectively (i.e., 1 weekafter the second boost with VEE-MAG25mer srRNA). The immune responsesmeasured 1 week after the second boost with VEE-MAG25mer srRNA (week 9)was ˜2-fold higher than that measured just before the boost immunization(FIG. 20D).

TABLE 22 Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for VEE-MAG25mer srRNA-LNP1(30 μg) (Group 1) Antigen Wk Env CL9 EnvTL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  1 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ±0  2 39.7 ± 22.7 35.4 ± 25.1 3.2 ± 3.6  33 ± 28.1 30.9 ± 20.3 28.3 ±17.5  3  2 ± 2.4 0.2 ± 1.8 1.8 ± 2.4 3.7 ± 1.9 1.7 ± 2.8 4.9 ± 2.3  4  1± 1.8 0.3 ± 1.2 5.5 ± 3.6 2.3 ± 2.2 5.7 ± 2.7 0.8 ± 0.8  5 0.5 ± 0.9 1.4± 3.8 3.1 ± 1.6 2.3 ± 2.7 1.9 ± 2  1.4 ± 1.2  6 1.9 ± 1.8 −0.3 ± 3   1.7 ± 1.2 1.4 ± 1.4 0.8 ± 1.1 1.1 ± 1   8 −0.4 ± 0.8  −0.9 ± 2.9  0.5 ±1.3  3 ± 1.1 2.2 ± 2.1 3.7 ± 2   9  1 ± 1.7 1.2 ± 4.2 7.2 ± 3.9 0.5 ±0.7 1.6 ± 3  3 ± 1 10 3.8 ± 1.8 11 ± 5  −1.1 ± 1.1  1.9 ± 0.9 1.3 ± 1.60.2 ± 0.5

TABLE 23 Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for VEE-MAG25mer srRNA-LNP1(100 μg) (Group 2) Antigen Wk Env CL9 EnvTL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  1 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ±0  2  7.9 ± 17.2 23.2 ± 17.4 11.4 ± 4.9  41.7 ± 16.5  15 ± 13.5 8.9 ±6.2  3 −3.1 ± 4.6  −7.2 ± 6.5  2.3 ± 2.3 −0.3 ± 2.7  2.7 ± 5.1 2.2 ± 1.4 4 1.9 ± 3.8 −6.2 ± 7.6  10.5 ± 4.1  1.2 ± 2.9 5.6 ± 4.9 1.1 ± 0.8  5−2.6 ± 7     −8 ± 5.9 1.5 ± 1.7 6.4 ± 2.3 0.7 ± 4.3 3.3 ± 1.3  6 6.3 ±6.3 4.4 ± 8.3 6.6 ± 4.4 5.2 ± 5.2 3.9 ± 5  10.8 ± 6.9   8 −3.6 ± 7.2 −6.8 ± 7.3  −0.8 ± 1.2  3.4 ± 4.2 6.4 ± 7.5 5.7 ± 2.7  9 8.1 ± 2.4 20.6± 23.4 18.9 ± 5.7  8.1 ± 8.9   9 ± 11.2  40 ± 17.6 10 3.1 ± 8  −3.9 ±8.5  3.3 ± 1.8 0.6 ± 2.9 7.4 ± 6.4 6.1 ± 2.5

TABLE 24 Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for VEE-MAG25mer srRNA-LNP2(100 μg) (Group 3) Antigen Wk Env CL9 EnvTL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  1 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ±0  2 −5.9 ± 3.8  −0.3 ± 0.5  −0.5 ± 1.5  −5.7 ± 6.1   −1 ± 1.3 −3.2 ±5.5   3 0.7 ± 5.2 3.4 ± 2.4 4.2 ± 4.6 18.3 ± 15.5 11.9 ± 5.1  −0.4 ±8.2   4 −3.8 ± 5.5  2.3 ± 1.8 11.3 ± 6.1  −3.1 ± 5.6  8.5 ± 4  −1.5 ±6.1   5 −3.7 ± 5.7  −0.1 ± 0.7  −0.2 ± 1.6  3.4 ± 8.5  3 ± 3.1 −4.6 ±5     6 12.3 ± 15  7.8 ± 4.9 24.7 ± 19.8 23.2 ± 22.5 18.7 ± 15.8 0.5 ±6.2  8  5.9 ± 12.3 −0.1 ± 0.7  −0.5 ± 1.3   8.8 ± 14.4 8.7 ± 8  −1.3 ±4     9 16.1 ± 13.4 16.5 ± 4   22.9 ± 4.2   13 ± 13.2 16.4 ± 7.8  19.6 ±9.2  10 29.9 ± 21.8  22 ± 19.5 0.5 ± 2.6 22.2 ± 22.6 35.3 ± 15.8 19.4 ±17.3

TABLE 25 Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for ChAdV68.5WTnt.MAG25mer prime Antigen Wk Env CL9 Env TL9 Gag CM9Gag LW9 Pol SV9 Tat TL8  1  178 ± 68.7 206.5 ± 94.8 221.2 ± 120  15.4 ±16.7  33.3 ± 25.9  563.5 ± 174.4  2 311.2 ± 165.5  278.8 ± 100.9 344.6 ±110.8 46.3 ± 13.5 181.6 ± 76.8  621.4 ± 220.9  3 277.3 ± 101.1 359.6 ±90.5 468.2 ± 106.6 41.7 ± 11.1 169.8 ± 57.8  549.4 ± 115.7  4  140 ±46.5 169.6 ± 46.8 239.4 ± 37   26.5 ± 11.4   75 ± 31.6 322.2 ± 50.7  5155.6 ± 62.1  406.7 ± 96.4 542.7 ± 143.3 35.1 ± 16.6 134.2 ± 53.7 538.5± 91.9  6 78.9 ± 42.5  95.5 ± 29.4 220.9 ± 75.3  −1.4 ± 5.3   43.4 ±19.6 308.1 ± 42.6  8 88.4 ± 30.4 162.1 ± 30.3 253.4 ± 78.6  21.4 ± 11.2 53.7 ± 22.3 217.8 ± 45.2  9 158.5 ± 69   322.3 ± 87.2 338.2 ± 137.1 5.6 ± 12.4 109.2 ± 17.9 314.8 ± 43.4 10 97.3 ± 32.5 133.2 ± 27  154.9 ±59.2  10 ± 6    26 ± 16.7 125.5 ± 27.7

Results

Mamu-A*01 Indian rhesus macaques were immunized withChAdV68.5-WTnt.MAG25mer. Antigen-specific cellular immune responses inperipheral blood mononuclear cells (PBMCs) were measured to sixdifferent Mamu-A*01 restricted epitopes prior to immunization and 4, 5,6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24weeks after the initial immunization (FIG. 21 and Table 27). Animalsreceived boost immunizations with VEE-MAG25mer srRNA using the LNP2formulation on weeks 4, 12, and 20. Combined antigen-specific immuneresponses of 1750, 4225, 1100, 2529, 3218, 1915, 1708, 1561, 5077, 4543,4920, 5820, 3395, 2728, 1996, 1465, 4730, 2984, 2828, or 3043 SFCs per10⁶ PBMCs (six epitopes combined) were measured 4, 5, 6, 7, 8, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks after theinitial immunization with ChAdV68.5WTnt.MAG25mer (FIG. 21 ). Immuneresponses measured 1 week after the second boost immunization (week 13)with VEE-MAG25mer srRNA were ˜3-fold higher than that measured justbefore the boost immunization (week 12). Immune responses measured 1week after the third boost immunization (week 21) with VEE-MAG25mersrRNA, were ˜3-fold higher than that measured just before the boostimmunization (week 20), similar to the response observed for the secondboost.

Mamu-A*01 Indian rhesus macaques were also immunized with VEE-MAG25mersrRNA using two different LNP formulations (LNP1 and LNP2).Antigen-specific cellular immune responses in peripheral bloodmononuclear cells (PBMCs) were measured to six different Mamu-A*01restricted epitopes prior to immunization and 4, 5, 6, 7, 8, 10, 11, 12,13, 14, or 15 weeks after the initial immunization (FIGS. 22 and 23 ,Tables 28 and 29). Animals received boost immunizations withVEE-MAG25mer srRNA using the respective LNP1 or LNP2 formulation onweeks 4 and 12. Combined antigen-specific immune responses of 168, 204,103, 126, 140, 145, 330, 203, and 162 SFCs per 106 PBMCs (six epitopescombined) were measured 4, 5, 7, 8, 10, 11, 13, 14, 15 weeks after theimmunization with VEE-MAG25mer srRNA-LNP2 (FIG. 22 ). Combinedantigen-specific immune responses of 189, 185, 349, 437, 492, 570, 233,886, 369, and 381 SFCs per 10⁶ PBMCs (six epitopes combined) weremeasured 4, 5, 7, 8, 10, 11, 12, 13, 14, 15 weeks after the immunizationwith VEE-MAG25mer srRNA-LNP1 (FIG. 23 ).

TABLE 27 Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for priming vaccination with ChAdV68.5WTnt.MAG25mer (Group 1)Antigen Wk Env CL9 Env TL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  4  173 ±41.6 373.5 ± 87.3 461.4 ± 74.2  38.4 ± 26.1 94.5 ± 26   609.2 ± 121.9  5412.7 ± 138.4  987.8 ± 283.3 1064.4 ± 266.9  85.6 ± 31.2  367.2 ± 135.21306.8 ± 332.8  6 116.2 ± 41.2  231.1 ± 46.3 268.3 ± 90.7 86.1 ± 42 174.3 ± 61  223.9 ± 38.1  7 287.4 ± 148.7  588.9 ± 173.9  693.2 ± 224.8 92.1 ± 33.5 172.9 ± 55.6  694.6 ± 194.8  8 325.4 ± 126.6 735.8 ± 212  948.9 ± 274.5 211.3 ± 62.7 179.1 ± 50   817.3 ± 185.2 10  312 ± 129.7 543.2 ± 188.4  618.6 ± 221.7 −5.7 ± 4.1 136.5 ± 51.3 309.9 ± 85.6 11248.5 ± 81.1   348.7 ± 129.8  581.1 ± 205.5 −3.1 ± 4.4  119 ± 51.2 413.7 ± 144.8 12 261.9 ± 68.2  329.9 ± 83   486.5 ± 118.6 −1.2 ± 5.1132.8 ± 31.8 350.9 ± 69.3 13 389.3 ± 167.7 1615.8 ± 418.3 1244.3 ± 403.6 1.3 ± 8.1 522.5 ± 155  1303.3 ± 385.6 14 406.3 ± 121.6  1616 ± 491.71142.3 ± 247.2  6.6 ± 11.1 322.7 ± 94.1 1048.6 ± 215.6 15 446.8 ± 138.71700.8 ± 469.1 1306.3 ± 294.4    43 ± 24.5 421.2 ± 87.9 1001.5 ± 236.416 686.8 ± 268.8 1979.5 ± 541.7 1616.8 ± 411.8  2.4 ± 7.8  381.9 ± 116.41152.8 ± 352.7 17 375.8 ± 109.3 1378.6 ± 561.2  773.1 ± 210.3 −1.4 ± 4.3177.6 ± 93.7 691.7 ± 245  18 255.9 ± 99.7  1538.4 ± 498.1  498.7 ± 152.3−5.3 ± 3.3  26.2 ± 13.4  413.9 ± 164.8 19  133 ± 62.6  955.9 ± 456.8 491.1 ± 121.8 −5.7 ± 4.1  50.3 ± 25.4  371.2 ± 123.7 20 163.7 ± 55.8  641.7 ± 313.5 357.9 ± 91.1  2.6 ± 7.5  41.4 ± 24.2 257.8 ± 68.9 21319.9 ± 160.5 2017.1 ± 419.9 1204.8 ± 335.2 −3.7 ± 5.1  268.1 ± 109.6924.1 ± 301  22 244.7 ± 105.6 1370.9 ± 563.5 780.3 ± 390  −3.6 ± 5.1118.2 ± 68.1  473.3 ± 249.3 23 176.7 ± 81.8  1263.7 ± 527.3  838.6 ±367.9 −5.7 ± 4.1 73.6 ± 49   480.9 ± 163.9 24 236.5 ± 92   1324.7 ±589.3 879.7 ± 321  −0.4 ± 5.7  104 ± 53.1   498 ± 135.8

TABLE 28 Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for priming vaccination with VEE-MAG25mer srRNA-LNP2 (300 μg) (Group2) Antigen Wk Env CL9 Env TL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  4  46 ±27.1 18.4 ± 6.8  58.3 ± 45.8 29.9 ± 20.8 4.9 ± 2.3 10.7 ± 4    5 85.4 ±54  5.2 ± 5.8 52.4 ± 51.2 34.5 ± 35  11.8 ± 12.2 14.4 ± 7.9   7 18.6 ±32.5 1.9 ± 1.7 59.4 ± 55.7  9.3 ± 10.7 3.3 ± 3  10.7 ± 6.1   8 36.6 ±39.4 6.3 ± 3.9 48.7 ± 39.9 13.5 ± 8.8  3.8 ± 3.6 17.2 ± 9.7  10 69.1 ±59.1 4.4 ± 1.9 39.3 ± 38  14.7 ± 10.8 4.4 ± 5.3 8.5 ± 5.3 11  43 ± 38.822.6 ± 21.1 30.2 ± 26.2 3.3 ± 2.2 5.8 ± 3.5 40.3 ± 25.5 13 120.4 ± 78.3 68.2 ± 43.9 54.2 ± 36.8 21.8 ± 7.4  17.7 ± 6.1  47.4 ± 27.3 14  76 ±44.8  28 ± 19.5 65.9 ± 64.3 −0.3 ± 1.3  2.5 ± 2  31.1 ± 26.5 15 58.9 ±41.4 19.5 ± 15.1 55.4 ± 51  2.5 ± 2  5.5 ± 3.6 20.1 ± 15.7

TABLE 29 Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for priming vaccination with VEE-MAG25mer srRNA-LNP1 (300 μg) (Group3) Antigen Wk Env CL9 Env TL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  4 19.5 ±8.7  13.3 ± 13.1 16.5 ± 15.3 10.5 ± 7.3  35.9 ± 24.8 92.9 ± 91.6  5 87.9± 43.9 12.7 ± 11.7 37.2 ± 31.9 21.1 ± 23.8 13.2 ± 13.7 12.6 ± 13.7  721.1 ± 13.3 48.8 ± 48.4 51.7 ± 39.5  9.1 ± 10.5 58.6 ± 55.8 159.4 ±159    8 47.7 ± 21.7 66.4 ± 52.2 59.8 ± 57.4 49.4 ± 28  79.4 ± 63  133.8 ± 132.1 10   49 ± 30.2 42.2 ± 41.1 139.3 ± 139.3 51.6 ± 51.2 78.2± 75.8 131.7 ± 131.6 11   42 ± 26.8 20.9 ± 21.4 177.1 ± 162   −6.3 ±4.3  104.3 ± 104.1 231.5 ± 230.1 12 40.2 ± 19   20.3 ± 11.9 42.2 ± 46.73.7 ± 6.7   57 ± 44.7   70 ± 69.2 13 81.2 ± 48.9 38.2 ± 37.6 259.4 ±222.2  −4 ± 4.1 164.1 ± 159.3 347.3 ± 343.5 14 34.5 ± 31.8  5.3 ± 11.6138.6 ± 137.3 −4.7 ± 5.2  52.3 ± 52.9 142.6 ± 142.6 15 49 ± 24 6.7 ± 9.8167.1 ± 163.8 −6.4 ± 4.2  47.8 ± 42.3 116.6 ± 114.5

srRNA Dose Ranging Study

In one implementation of the present invention, an srRNA dose rangingstudy can be conducted in Mamu A01 Indian rhesus macaques to identifywhich srRNA dose to progress to NHP immunogenicity studies. In oneexample, Mamu A01 Indian rhesus macaques can be administered with ansrRNA vector encoding model antigens that includes multiple Mamu A01restricted epitopes by IM injection. In another example, an anti-CTLA-4monoclonal antibody can be administered SC proximal to the site of IMvaccine injection to target the vaccine draining lymph node in one groupof animals. PBMCs can be collected every 2 weeks after the initialvaccination for immune monitoring. The study arms are described in below(Table 30).

TABLE 30 Non-GLP RNA dose ranging study in Indian Rhesus Macaques GroupPrime Boost 1 Boost 2 1 srRNA-LNP srRNA-LNP srRNA-LNP (Low Dose) (LowDose) (Low Dose) 2 srRNA-LNP srRNA-LNP srRNA-LNP (Mid Dose) (Mid Dose)(Mid Dose) 3 srRNA-LNP srRNA-LNP srRNA-LNP (High Dose) (High Dose) (HighDose) 4 srRNA-LNP srRNA-LNP srRNA-LNP (High Dose) + (High Dose) + (HighDose) + anti-CTLA-4 anti-CTLA-4 anti-CTLA-4 * Dose range of srRNA to bedetermined with the high dose ≤300 □g.

Immunogenicity Study in Indian Rhesus Macaques

Vaccine studies were conducted in Mamu A01 Indian rhesus macaques (NHPs)to demonstrate immunogenicity using the antigen vectors. FIG. 34illustrates the vaccination strategy. Three groups of NHPs wereimmunized with ChAdV68.5-WTnt.MAG25mer and either with the checkpointinhibitor anti-CTLA-4 antibody Ipilimumab (Groups 5 & 6) or without thecheckpoint inhibitor (Group 4). The antibody was administered eitherintra-venously (group 5) or subcutaneously (group 6). Triangles indicateChAdV68 vaccination (1e12 vp/animal) at weeks 0 & 32. Circles representalphavirus vaccination at weeks 0, 4, 12, 20, 28 and 32.

The time course of CD8+ anti-epitope responses in the immunized NHPs arepresented for ChAdV-MAG immunization alone (FIG. 35 and Table 31A),ChAdV-MAG immunization with the checkpoint inhibitor delivered IV (FIG.36 and Table 31B), and ChAdV-MAG immunization with the checkpointinhibitor delivered SC (FIG. 37 and Table 31C). The results demonstrateChAdV68 vectors efficiently primed CD8+ responses in primates,alphavirus vectors efficiently boosted the ChAdV68 vaccine primingresponse, checkpoint inhibitor whether delivered IV or SC amplified bothpriming and boosting responses, and ChAdV vectors readministered postvaccination to effectively boosted the immune responses.

TABLE 31A CD8+ anti-epitope responses in Rhesus Macaques dosed withchAd-MAG (Group 4). Mean SFC/1e6 splenocytes +/− the standard error isshown Antigen Wk Env CL9 Env TL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  4  173± 41.6 373.5 ± 87.3  461.4 ± 74.2  38.4 ± 26.1 94.5 ± 26   609.2 ± 121.9 5  412.7 ± 138.4 987.8 ± 283.3 1064.4 ± 266.9  85.6 ± 31.2 367.2 ±135.2 1306.8 ± 332.8   6 116.2 ± 41.2 231.1 ± 46.3  268.3 ± 90.7  86.1 ±42   174.3 ± 61   223.9 ± 38.1   7  287.4 ± 148.7 588.9 ± 173.9 693.2 ±224.8 92.1 ± 33.5 172.9 ± 55.6  694.6 ± 194.8  8  325.4 ± 126.6 735.8 ±212   948.9 ± 274.5 211.3 ± 62.7  179.1 ± 50   817.3 ± 185.2 10   312 ±129.7 543.2 ± 188.4 618.6 ± 221.7 −5.7 ± 4.1  136.5 ± 51.3  309.9 ±85.6  11 248.5 ± 81.1 348.7 ± 129.8 581.1 ± 205.5 −3.1 ± 4.4   119 ±51.2 413.7 ± 144.8 12 261.9 ± 68.2 329.9 ± 83   486.5 ± 118.6 −1.2 ±5.1  132.8 ± 31.8  350.9 ± 69.3  13  389.3 ± 167.7 1615.8 ± 418.3 1244.3 ± 403.6  1.3 ± 8.1 522.5 ± 155   1303.3 ± 385.6  14  406.3 ±121.6  1616 ± 491.7 1142.3 ± 247.2   6.6 ± 11.1 322.7 ± 94.1  1048.6 ±215.6  15  446.8 ± 138.7 1700.8 ± 469.1  1306.3 ± 294.4    43 ± 24.5421.2 ± 87.9  1001.5 ± 236.4  16  686.8 ± 268.8 1979.5 ± 541.7  1616.8 ±411.8  2.4 ± 7.8 381.9 ± 116.4 1152.8 ± 352.7  17  375.8 ± 109.3 1378.6± 561.2  773.1 ± 210.3 −1.4 ± 4.3  177.6 ± 93.7  691.7 ± 245   18 255.9± 99.7 1538.4 ± 498.1  498.7 ± 152.3 −5.3 ± 3.3  26.2 ± 13.4 413.9 ±164.8 19  133 ± 62.6 955.9 ± 456.8 491.1 ± 121.8 −5.7 ± 4.1  50.3 ± 25.4371.2 ± 123.7 20 163.7 ± 55.8 641.7 ± 313.5 357.9 ± 91.1  2.6 ± 7.5 41.4± 24.2 257.8 ± 68.9  21  319.9 ± 160.5 2017.1 ± 419.9  1204.8 ± 335.2 −3.7 ± 5.1  268.1 ± 109.6 924.1 ± 301   22  244.7 ± 105.6 1370.9 ±563.5  780.3 ± 390   −3.6 ± 5.1  118.2 ± 68.1  473.3 ± 249.3 23 176.7 ±81.8 1263.7 ± 527.3  838.6 ± 367.9 −5.7 ± 4.1  73.6 ± 49   480.9 ± 163.924 236.5 ± 92  1324.7 ± 589.3  879.7 ± 321   −0.4 ± 5.7   104 ± 53.1  498 ± 135.8 25 136.4 ± 52.6 1207.1 ± 501.6    924 ± 358.5  6.2 ± 10.574.1 ± 34.4 484.6 ± 116.7 26  278.2 ± 114.4  1645 ± 661.7 1170.2 ±469.9  −2.9 ± 5.7  80.6 ± 55.8 784.4 ± 214.1 27  159 ± 56.8 961.7 ±547.1 783.6 ± 366.4  −5 ± 4.3 63.6 ± 27.5 402.9 ± 123.4 28 189.6 ± 75.71073.1 ± 508.8  668.3 ± 312.5 −5.7 ± 4.1  80.3 ± 38.3 386.4 ± 122   29155.3 ± 69.1 1102.9 ± 606.1  632.9 ± 235   34.5 ± 24.2   80 ± 35.5 422.5± 122.9 30 160.2 ± 59.9   859 ± 440.9   455 ± 209.1  −3 ± 5.3 60.5 ±28.4 302.7 ± 123.2 31 122.2 ± 49.7 771.1 ± 392.7 582.2 ± 233.5 −5.7 ±4.1  55.1 ± 27.3 295.2 ± 68.3  32 119.3 ± 28.3 619.4 ± 189.7   566 ±222.1 −3.7 ± 5.1  21.9 ± 4.5  320.5 ± 76.4  33 380.5 ± 122  1636.1 ±391.4  1056.2 ± 205.7  −5.7 ± 4.1  154.5 ± 38.5  988.4 ± 287.7 34 1410.8± 505.4 972.4 ± 301.5 319.6 ± 89.6  −4.8 ± 4.2  141.1 ± 49.8  1375.5 ±296.7  37 130.8 ± 29.2   500 ± 156.9 424.9 ± 148.9 −3.5 ± 4.7  77.7 ±24.6 207.1 ± 42.4  38 167.7 ± 54.8 1390.8 ± 504.7  830.4 ± 329.1 −5.5 ±4.1  111.8 ± 43.2    516 ± 121.7

TABLE 31B CD8+ anti-epitope responses in Rhesus Macaques dosed withChAdV-MAG plus anti-CTLA4 antibody (Ipilimumab) delivered IV (Group 5).Mean SFC/1e6 splenocytes +/− the standard error is shown Antigen Wk EnvCL9 Env TL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  4 1848.1 ± 432.2 1295.7 ±479.7 1709.8 ± 416.9 513.7 ± 219.8  838.5 ± 221.1 2514.6 ± 246.5  51844.1 ± 410.2 2367.5 ± 334.4 1983.1 ± 370.7 732.1 ± 249.4 1429.7 ±275.3 2517.7 ± 286.5  6  822.4 ± 216.7 1131.2 ± 194.7  796.8 ± 185.8226.8 ± 70    802.2 ± 101.4  913.5 ± 222.7  7 1147.2 ± 332.9  1066 ±311.2 1149.8 ± 467.3 267.4 ± 162.6  621.5 ± 283.2 1552.2 ± 395.1  81192.7 ± 188.8 1461.5 ± 237.7 1566.9 ± 310.5 522.5 ± 118.6  662.3 ±142.4  1706 ± 216.7 10  1249 ± 220.3 1170.6 ± 227.7 1297.3 ± 264.7 −0.3± 4.4  551.8 ± 90.5 1425.3 ± 142.6 11  934.2 ± 221.7   808 ± 191.31003.1 ± 293.4 1.9 ± 4.3 364.2 ± 76.6 1270.8 ± 191.6 12 1106.2 ± 216.6 896.7 ± 190.7 1020.1 ± 243.3 1.3 ± 3.9 436.6 ± 90   1222 ± 155.4 132023.8 ± 556.3 3696.7 ± 1.7  2248.5 ± 436.4 −4.5 ± 3.5   2614 ± 406.13700 ± 0  14 1278.7 ± 240  2639.5 ± 387  1654.6 ± 381.1  −6 ± 2.1  988.8± 197.9 2288.3 ± 298.7 15 1458.9 ± 281.8 2932.5 ± 488.7 1893.4 ± 499 74.6 ± 15.6 1657.8 ± 508.9 2709.1 ± 428.7 16 1556.8 ± 243  2143.8 ±295.2 2082.8 ± 234.2 −5.8 ± 2.5  1014.6 ± 161.4 2063.7 ± 86.7  17  1527± 495.1  2213 ± 677.1 1767.7 ± 391.8 15.1 ± 5.9   633.8 ± 133.9 2890.8 ±433.9 18 1068.2 ± 279.9 1940.9 ± 204.1 1114.1 ± 216.1 −5.8 ± 2.5  396.6± 77.6 1659.4 ± 171.7 19  760.7 ± 362.2 1099.5 ± 438.4  802.7 ± 192.5−2.4 ± 3.3  262.2 ± 62.2 1118.6 ± 224.2 20  696.3 ± 138.2 954.9 ± 198  765.1 ± 248.4 −1.4 ± 4.4  279.6 ± 89.3  1139 ± 204.5 21 1201.4 ± 327.93096 ± 1.9   1901 ± 412.1 −5.8 ± 2.5  1676.3 ± 311.5 2809.3 ± 195.8 221442.5 ± 508.3 2944.7 ± 438.6 1528.4 ± 349.6 2.8 ± 5.1  940.7 ± 160.52306.3 ± 218.6 23 1400.4 ± 502.2 2757.1 ± 452.9 1604.2 ± 450.1 −5.1 ±2.3   708.1 ± 162.6 2100.4 ± 362.9 24  1351 ± 585.1 2264.5 ± 496  1080.6± 253.8 0.3 ± 6.5  444.2 ± 126.4 1823.7 ± 306.5 25 1211.5 ± 505.2 2160.4± 581.8  970.8 ± 235.9 2.5 ± 3.8  450.4 ± 126.9 1626.2 ± 261.3 26  1443± 492.5 2485 ± 588 1252.5 ± 326.4 −0.2 ± 6    360.2 ± 92.3 2081.9 ±331.1 27  896.2 ± 413.3  1686 ± 559.5   751 ± 192.1 −3.7 ± 2.5  247.4 ±82.8 1364.1 ± 232  28 1147.8 ± 456.9 1912.1 ± 417.1  930.3 ± 211.4 −5.8± 2.5  423.9 ± 79.6 1649.3 ± 315  29 1038.5 ± 431.9 1915.2 ± 626.1 786.8 ± 205.9 23.5 ± 8.3  462.8 ± 64  1441.5 ± 249.7 30  730.5 ± 259.31078.6 ± 211.5  699.1 ± 156.2 −4.4 ± 2.7  234.4 ± 43.9 1160.6 ± 112.6 31 750.4 ± 328.3  1431 ± 549.9  650.6 ± 141.1 −5.2 ± 3    243.4 ± 56.4 868.9 ± 142.8 32  581.4 ± 227.7 1326.6 ± 505.2 573.3 ± 138  −3.2 ± 4.2 160.8 ± 49.2  936.4 ± 110.4 33 2198.4 ± 403.8 3093.4 ± 123.3 2391.8 ±378.4 7.1 ± 8.5 1598.1 ± 343.1 2827.5 ± 289.5 34 2654.3 ± 337  2709.9 ±204.3 1297.5 ± 291.4 0.4 ± 4.2 1091.8 ± 242.9  1924 ± 245.7 37  846.8 ±301.7 1706.9 ± 196   973.6 ± 149.3 50.5 ± 45.2  777.3 ± 140.2 1478.8 ±94.3 

TABLE 31C CD8+ anti-epitope responses in Rhesus Macaques dosed withchAd-MAG plus anti-CTLA4 antibody (Ipilimumab) delivered SC (Group 6).Mean SFC/1e6 splenocytes +/− the standard error is shown Antigen Wk EnvCL9 Env TL9 Gag CM9 Gag LW9 Pol SV9 Tat TL8  4 598.3 ± 157.4  923.7 ±306.8 1075.6 ± 171.8 180.5 ± 74.1 752.3 ± 245.8 1955.3 ± 444.4  5 842.2± 188.5 1703.7 ± 514.2 1595.8 ± 348.4 352.7 ± 92.3 1598.9 ± 416.8 2163.7 ± 522.1  6 396.4 ± 45.3   728.3 ± 232.7  503.8 ± 151.9 282 ± 69463.1 ± 135.7  555.2 ± 191.5  7 584.2 ± 177   838.3 ± 254.9 1013.9 ±349.4 173.6 ± 64.3 507.4 ± 165.7 1222.8 ± 368   8 642.9 ± 134  1128.6 ±240.6 1259.1 ± 163.8 366.1 ± 72.8 726.7 ± 220.9 1695.6 ± 359.4 10 660.4± 211.4  746.9 ± 222.7  944.8 ± 210.2 −1.2 ± 1.9 523.4 ± 230.7  787.3 ±308.3 11 571.2 ± 162   609.4 ± 194.3  937.9 ± 186.5 −8.9 ± 2.3 511.6 ±229.6 1033.3 ± 315.7 12 485.3 ± 123.7  489.4 ± 142.7  919.3 ± 214.1 −8.9± 2.3 341.6 ± 139.4 1394.7 ± 432.1 13 986.9 ± 154.5 2811.9 ± 411.31687.7 ± 344.3 −4.1 ± 5.1 1368.5 ± 294.2   2751 ± 501.9 14 945.9 ± 251.42027.7 ± 492.8 1386.7 ± 326.7 −5.7 ± 2.8 708.9 ± 277.1 1588.2 ± 440.1 151075.2 ± 322.4   2386 ± 580.7 1606.3 ± 368.1 −5.4 ± 3.2 763.3 ± 248.81896.5 ± 507.8 16 1171.8 ± 341.6  2255.1 ± 439.6 1672.2 ± 342.3 −7.8 ±2.4 1031.6 ± 228.8  1896.4 ± 419.9 17 1118.2 ± 415.4  2156.3 ± 476 1345.3 ± 377.7 −1.1 ± 6.7 573.7 ± 118.8 1614.4 ± 382.3 18 861.3 ± 313.82668.2 ± 366.8 1157.2 ± 259.6 −8.9 ± 2.3 481.2 ± 164   1725.8 ± 511.4 19719.2 ± 294.2 1447.2 ± 285    968 ± 294.5 −2.2 ± 4.6 395.6 ± 106.11199.6 ± 289.2 20 651.6 ± 184  1189.8 ± 242.8  947.4 ± 249.8 −8.9 ± 2.3  355 ± 106.3 1234.7 ± 361.7 21 810.3 ± 301.9 2576.2 ± 283.7  1334 ±363.1 −8.9 ± 2.3 892.2 ± 305   1904.4 ± 448.1 22  775 ± 196.4  2949 ±409.7 1421.8 ± 309.7    38 ± 27.8   577 ± 144.2 2330.6 ± 572.3 23 584.9± 240.2 1977.9 ± 361.4 1209.8 ± 405.1 −7.3 ± 3.2 273.7 ± 93.3  1430.6 ±363.9 24 485.4 ± 194.4 1819.8 ± 325.5  837.2 ± 261.4 −3.4 ± 4.1 234.4 ±71.1   943.9 ± 243.3 25 452.3 ± 175   2072 ± 405.7  957.1 ± 293.1 −8.9 ±2.3  163 ± 43.2 1341.2 ± 394.7 26 517.9 ± 179.1  2616 ± 567.5 1126.6 ±289  −8.3 ± 2.3 199.9 ± 89.2  1615.7 ± 385.6 27 592.8 ± 171.7 1838.3 ±372.4  749.3 ± 170.4 −7.3 ± 2.5 325.5 ± 98.7  1110.7 ± 308.8 28  793 ±228.5 1795.4 ± 332.3 1068.7 ± 210.3  2.5 ± 4.1 553.1 ± 144.3 1480.8 ±357.1 29 661.8 ± 199.9 2140.6 ± 599.3 1202.7 ± 292.2 −8.7 ± 2.8 558.9 ±279.2 1424.2 ± 408.6 30 529.1 ± 163.3 1528.2 ± 249.8  840.5 ± 218.3 −8.9± 2.3 357.7 ± 149.4 1029.3 ± 335  31 464.8 ± 152.9 1332.2 ± 322.7  726.3± 194.3 −8.9 ± 2.3 354.3 ± 158.6 884.4 ± 282  32 612.9 ± 175.3 1584.2 ±390.2 1058.3 ± 219.8 −8.7 ± 2.8 364.6 ± 149.8 1388.8 ± 467.3 33 1600.2 ±416.7  2597.4 ± 367.9 2086.4 ± 414.8 −6.3 ± 3.3 893.8 ± 266   2490.6 ±416.4 34 2814.6 ± 376.2  2713.6 ± 380.8 1888.8 ± 499.4 −7.5 ± 3.1 1288.9± 438.9  2428.1 ± 458.9 37 1245.9 ± 471.7  1877.7 ± 291.2 1606.6 ± 441.914.2 ± 13  1227.5 ± 348.1  1260.7 ± 342 

Memory Phenotyping in Indian Rhesus Macaques

Rhesus macaque were immunized with ChAdV68.5WTnt.MAG25mer/VEE-MAG25mersrRNA heterologous prime/boost regimen with or without anti-CTLA4, andboosted again with ChAdV68.5WTnt.MAG25mer. Groups were assessed 11months after the final ChAdV68 administration (study month 18). byELISpot was performed as described. FIG. 38 and Table 38 shows cellularresponses to six different Mamu-A*01 restricted epitopes as measured byELISpot both pre-immunization (left panel) and after 18 months (rightpanel). The detection of responses to the restricted epitopesdemonstrates antigen-specific memory responses were generated byChAdV68/samRNA vaccine protocol.

To assess memory, CD8+ T-cells recognizing 4 different rhesus macaqueMamu-A*01 class I epitopes encoded in the vaccines were monitored usingdual-color Mamu-A*01 tetramer labeling, with each antigen beingrepresented by a unique double positive combination, and allowed theidentification of all 4 antigen-specific populations within a singlesample. Memory cell phenotyping was performed by co-staining with thecell surface markers CD45RA and CCR7. FIG. 39 and Table 39 shows theresults of the combinatorial tetramer staining and CD45RA/CCR7co-staining for memory T-cells recognizing four different Mamu-A*01restricted epitopes. The T cell phenotypes were also assessed by flowcytometry. FIG. 40 shows the distribution of memory cell types withinthe sum of the four Mamu-A*01 tetramer+CD8+ T-cell populations at studymonth 18. Memory cells were characterized as follows:CD45RA+CCR7+=naïve, CD45RA+CCR7-=effector (Teff), CD45RA-CCR7+=centralmemory (Tcm), CD45RA-CCR7-=effector memory (Tem). Collectively, theresults demonstrate that memory responses were detected at least oneyear following the last boost indicating long lasting immunity,including effector, central memory, and effector memory populations.

TABLE 38 Mean spot forming cells (SFC) per 10⁶ PBMCs for each animal atboth pre-prime and memory assessment time points (18 months). Pre-primebaseline 18 months Tat Gag Env Env Gag Pol Tat Gag Env Env Gag PolAnimal TL8 CM9 TL9 CL9 LW9 SV9 TL8 CM9 TL9 CL9 LW9 SV9 1 1.7 0.0 0.0 5.00.0 13.7 683.0 499.2 1100.3 217.5 47.7 205.3 2 0.0 0.0 0.0 0.2 0.1 0.0773.4 ND 1500.0 509.3 134.5 242.5 3 0.0 0.0 6.7 6.8 10.2 3.3 746.3 167.5494.1 402.8 140.6 376.0 4 0.0 0.0 0.0 0.0 0.0 0.0 47.6 1023.9 85.1 44.244.2 47.6 5 15.3 6.7 18.6 15.6 5.2 12.1 842.4 467.7 1500.0 805.9 527.8201.8 6 3.1 0.0 0.0 15.5 6.9 5.3 224.3 720.3 849.0 296.9 32.4 121.9 ND =not determined due to technical exclusion

TABLE 39 Percent Mamu-A*01 tetramer positive out of live CD8+ cellsAnimal Tat 118 Gag CM9 Env 119 Env CL9 1 0.42 0.11 0.19 0.013 2 0.360.048 0.033 0.00834 3 0.97 0.051 0.35 0.048 4 0.46 0.083 0.17 0.028 50.77 0.45 0.14 0.2 6 0.71 0.16 0.17 0.04

Non-GLP RNA Dose Ranging Study (Higher Doses) in Indian Rhesus Macaques

This study was designed to (a) evaluate the immunogenicity ofVEE-MAG25mer srRNA at a dose of 300 μg as a homologous prime/boost orheterologous prime/boost in combination with ChAdV68.5WTnt.MAG25mer; (b)compare the immune responses of VEE-MAG25mer srRNA in lipidnanoparticles using LNP1 versus LNP2 at the 300 μg dose; and (c)evaluate the kinetics of T-cell responses to VEE-MAG25mer srRNA andChAdV68.5WTnt.MAG25mer immunizations.

The study arm was conducted in Mamu-A*01 Indian rhesus macaques todemonstrate immunogenicity. Vaccine immunogenicity in nonhuman primatespecies, such as Rhesus, is the best predictor of vaccine potency inhumans. Furthermore, select antigens used in this study are onlyrecognized in Rhesus macaques, specifically those with a Mamu-A*01 MHCclass I haplotype. Mamu-A*01 Indian rhesus macaques were randomized tothe different study arms (6 macaques per group) and administered an IMinjection bilaterally with either ChAdV68.5-WTnt.MAG25mer orVEE-MAG25mer srRNA encoding model antigens that includes multipleMamu-A*01 restricted antigens. The study arms were as described below.

PBMCs were collected prior to immunization and 4, 5, 6, 7, 8, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks after theinitial immunization for immune monitoring for group 1 (heterologousprime/boost). PBMCs were collected prior to immunization and 4, 5, 7, 8,10, 11, 12, 13, 14, or 15 weeks after the initial immunization forimmune monitoring for groups 2 and 3 (homologous prime/boost).

TABLE 26 Non-GLP immunogenicity study in Indian Rhesus Macaques GroupPrime Boost 1 Boost 2 Boost 3 1 ChAdV68.5WTnt VEE- VEE- VEE- .MAG25merMAG25mer MAG25mer MAG25mer srRNA-LNP2 srRNA-LNP2 srRNA-LNP2 (300 μg)(300 μg) (300 μg) 2 VEE- VEE- VEE- MAG25mer MAG25mer MAG25mer srRNA-LNP2srRNA-LNP2 srRNA-LNP2 (300 μg) (300 μg) (300 μg) 3 VEE- VEE- VEE-MAG25mer MAG25mer MAG25mer srRNA-LNP1 srRNA-LNP1 srRNA-LNP1 (300 μg)(300 μg) (300 μg)

XIX. Identification of MHC/Peptide Target-Reactive T Cells and TCRs

Target reactive T cells and TCRs are identified for one or more of theantigen/HLA peptides pairs, including any antigens described herein,such as tumor-associated antigens or infectious disease associatedantigens.

T cells can be isolated from blood, lymph nodes, or tumors of patients.T cells can be enriched for antigen-specific T cells, e.g., by sortingantigen -MHC tetramer binding cells or by sorting activated cellsstimulated in an in vitro co-culture of T cells and antigen-pulsedantigen presenting cells. Various reagents are known in the art forantigen-specific T cell identification including antigen-loadedtetramers and other MHC-based reagents.

Antigen-relevant alpha-beta (or gamma-delta) TCR dimers can beidentified by single cell sequencing of TCRs of antigen-specific Tcells. Alternatively, bulk TCR sequencing of antigen-specific T cellscan be performed and alpha-beta pairs with a high probability ofmatching can be determined using a TCR pairing method known in the art.

Alternatively or in addition, antigen-specific T cells can be obtainedthrough in vitro priming of naïve T cells from healthy donors. T cellsobtained from PBMCs, lymph nodes, or cord blood can be repeatedlystimulated by antigen-pulsed antigen presenting cells to primedifferentiation of antigen-experienced T cells. TCRs can then beidentified similarly as described above for antigen-specific T cellsfrom patients.

XX. E4 Deletion in ChAdV68 Vectors Demonstrates Improved Productivity

Clones of a ChAdV68 adenoviral vector was selected for improved virusproductivity. Fast growing/fit ChAdV68 viruses that express the modelTSNA cassette MAG were selected for during plaque isolation and analyzedas described below.

Materials and Methods

ChAdV68 Plaque Isolation

Serial dilutions (from 10⁻² to 10⁻⁹) of ChAdV68-MAG viruses were madeand 100 uL plated on HEK293A (ThermoFisher cat. no. R70507) cells seededat 1e6 cells/60 mM plate. 24 h post infection the media was removed andthe infected cells were overlaid with DMEM/1.25% agarose and plaqueswere allowed to grow for 10-15 days. During this time, 72 viral plaqueswere picked. The virus was eluted overnight in 0.5 mL of DMEM/5% FBSmedia and half of the elution (0.25 mL) was used to re-infect 293A cellsseeded at 1e5 cells/well of a 24 well plate. The viruses were amplifiedand infected onto 293A cells. Rapidly growing clones were selected forvirus production in 400 mL 293F (ThermoFisher cat. No. A14528)suspension cultures. The virus was purified by 2×CsCl gradientpurification and formulated into ARM buffer (25 mM NaCl, 20 mm Tris pH8.0, 2.5% Glycerol) by 3 rounds of dialysis. Viral particle titers weredetermined by Absorbance at 260 nm measurement post 0.1% SDS lysis at 56C. Infectious titers were determined using an anti-capsid immunostainingassay.

Next Generation Sequencing

DNA was purified from the purified virus using the QiAmp viral DNA kit(Qiagen) and subjected to NGS using the Illumina platform.

MOI Evaluation of Clone Productivity

Controlled infections were set up using the purified virus at an MOI of0.1 IU and incubated for 96 h. Infectious units were measured in celllysates. Production was compared to a non-plaque selected virus (pool).

Immunizations

Balb/c female mice were injected with 1×10⁹ or 1×10¹⁰ viral particles(VP) of ChAdV68.5WTnt.MAG25mer (SEQ ID NO:2) or ChAdV68-MAG-E4deleted(SEQ ID NO: 57; “MAG E4 Delta” and “ChAdV68-MAG-E4”) in 100 uL volume,bilateral intramuscular injection (50 uL per leg).

Mamu-A*01 Indian rhesus macaques were immunized as bilateralintramuscular injections into the quadriceps muscle with 1×10¹² viralparticles (5×10¹¹ viral particles per injection) ofChAdV68.5WTnt.MAG25mer (“ChAdV68-CMV-MAG”; SEQ ID NO:2; no E4 deletionor TET response element) or ChAdV68-E4d-CMT-MAG (SEQ ID NO:71; E4deletion and CMT TET response element [see below]). Macaques were alsoadministered 50 mg of an anti-CTLA4 antibody (Ipilimumab) SC on the dayof injection.

ChAdV68-E4d-CMT-MAG;  SEQ ID NO: 71CATCATCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATGACATTGATTATTGACTAGTTGTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGCCACCATGGCCGGGATGTTCCAGGCACTGTCCGAAGGCTGCACACCCTATGATATTAACCAGATGCTGAATGTCCTGGGAGACCACCAGGTCTCTGGCCTGGAGCAGCTGGAGAGCATCATCAACTTCGAGAAGCTGACCGAGTGGACAAGCTCCAATGTGATGCCTATCCTGTCCCCACTGACCAAGGGCATCCTGGGCTTCGTGTTTACCCTGACAGTGCCTTCTGAGCGGGGCCTGTCTTGCATCAGCGAGGCAGACGCAACCACACCAGAGTCCGCCAATCTGGGCGAGGAGATCCTGTCTCAGCTGTACCTGTGGCCCCGGGTGACATATCACTCCCCTTCTTACGCCTATCACCAGTTCGAGCGGAGAGCCAAGTACAAGAGACACTTCCCAGGCTTTGGCCAGTCTCTGCTGTTCGGCTACCCCGTGTACGTGTTCGGCGATTGCGTGCAGGGCGACTGGGATGCCATCCGGTTTAGATACTGCGCACCACCTGGATATGCACTGCTGAGGTGTAACGACACCAATTATTCCGCCCTGCTGGCAGTGGGCGCCCTGGAGGGCCCTCGCAATCAGGATTGGCTGGGCGTGCCAAGGCAGCTGGTGACACGCATGCAGGCCATCCAGAACGCAGGCCTGTGCACCCTGGTGGCAATGCTGGAGGAGACAATCTTCTGGCTGCAGGCCTTTCTGATGGCCCTGACCGACAGCGGCCCCAAGACAAACATCATCGTGGATTCCCAGTACGTGATGGGCATCTCCAAGCCTTCTTTCCAGGAGTTTGTGGACTGGGAGAACGTGAGCCCAGAGCTGAATTCCACCGATCAGCCATTCTGGCAGGCAGGAATCCTGGCAAGGAACCTGGTGCCTATGGTGGCCACAGTGCAGGGCCAGAATCTGAAGTACCAGGGCCAGAGCCTGGTCATCAGCGCCTCCATCATCGTGTTTAACCTGCTGGAGCTGGAGGGCGACTATCGGGACGATGGCAACGTGTGGGTGCACACCCCACTGAGCCCCAGAACACTGAACGCCTGGGTGAAGGCCGTGGAGGAGAAGAAGGGCATCCCAGTGCACCTGGAGCTGGCCTCCATGACCAATATGGAGCTGATGTCTAGCATCGTGCACCAGCAGGTGAGGACATACGGACCCGTGTTCATGTGCCTGGGAGGCCTGCTGACCATGGTGGCAGGAGCCGTGTGGCTGACAGTGCGGGTGCTGGAGCTGTTCAGAGCCGCCCAGCTGGCCAACGATGTGGTGCTGCAGATCATGGAGCTGTGCGGAGCAGCCTTTCGCCAGGTGTGCCACACCACAGTGCCATGGCCCAATGCCTCCCTGACCCCCAAGTGGAACAATGAGACAACACAGCCTCAGATCGCCAACTGTAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTATAGCGTGAGGGATACCCTGTGGCCCCGCGTGACATACCACATGAATAAGTACGCCTATCACATGCTGGAGAGGCGCGCCAAGTATAAGAGAGGCCCTGGCCCAGGCGCAAAGTTTGTGGCAGCATGGACCCTGAAGGCCGCCGCCGGCCCCGGCCCCGGCCAGTATATCAAGGCTAACAGTAAGTTCATTGGAATCACAGAGCTGGGACCCGGACCTGGATAATGAGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTTCCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCGGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCCGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCTGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCAATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATG

Immune Monitoring Mice

Lymphocytes were isolated from freshly harvested spleens and lymph nodesof immunized mice. Tissues were dissociated in RPMI containing 10% fetalbovine serum with penicillin and streptomycin (complete RPMI) using theGentleMACS tissue dissociator according to the manufacturer'sinstructions. Freshly isolated lymphocytes at a density of 2-5×10⁶cells/mL were incubated with 10 uM of the indicated peptides for 2hours. After two hours, brefeldin A was added to a concentration of 5ug/ml and cells were incubated with stimulant for an additional 4 hours.Following stimulation, viable cells were labeled with fixable viabilitydye eFluor780 according to manufacturer's protocol and stained withanti-CD8 APC (clone 53-6.7, BioLegend) at 1:400 dilution. Anti-IFNg PE(clone XMG1.2, BioLegend) was used at 1:100 for intracellular staining.Samples were collected on an Attune NxT Flow Cytometer (ThermoScientific). Flow cytometry data was plotted and analyzed using FlowJo.To assess degree of antigen-specific response, both the percent IFNg+ ofCD8+ cells and the total IFNg+ cell number/1×10⁶ live cells werecalculated in response to each peptide stimulant.

Immune Monitoring NHPs

PBMCs were isolated at indicated times after prime vaccination usingLymphocyte Separation Medium (LSM, MP Biomedicals) and LeucoSepseparation tubes (Greiner Bio-One) and resuspended in RPMI containing10% FBS and penicillin/streptomycin. Cells were counted on the AttuneNxT flow cytometer (Thermo Fisher) using propidium iodide staining toexclude dead and apoptotic cells. Cell were then adjusted to theappropriate concentration of live cells for subsequent analysis. Foreach monkey in the studies, T cell responses were measured using ELISpotor flow cytometry methods. T cell responses to 6 different rhesusmacaque Mamu-A*01 class I epitopes encoded in the vaccines weremonitored from PBMCs by measuring induction of cytokines, such asIFN-gamma, using ex vivo enzyme-linked immunospot (ELISpot) analysis.ELISpot analysis was performed according to ELISPOT harmonizationguidelines {DOI: 10.1038/nprot.2015.068} with the monkey IFNgELISpotPLUS kit (MABTECH). 200,000 PBMCs were incubated with 10 uM ofthe indicated peptides for 16 hours in 96-well IFNg antibody coatedplates. Spots were developed using alkaline phosphatase. The reactionwas timed for 10 minutes and was terminated by running plate under tapwater. Spots were counted using an AID vSpot Reader Spectrum. ForELISPOT analysis, wells with saturation >50% were recorded as “toonumerous to count”. Samples with deviation of replicate wells >10% wereexcluded from analysis. Spot counts were then corrected for wellconfluency using the formula: spot count+2×(spot count×%confluence/[100%−% confluence]). Negative background was corrected bysubtraction of spot counts in the negative peptide stimulation wellsfrom the antigen stimulated wells. Finally, wells labeled too numerousto count were set to the highest observed corrected value, rounded up tothe nearest hundred.

Results

Fast growing/fit ChAdV68 viruses that express the model TSNA cassetteMAG (ChAdV68.5WTnt.MAG25mer; SEQ ID NO:2) were selected for duringplaque isolation, as described. Of the original 75 plaques, 33 producedvirus, as indicated by some signs of CPE (Cytopathic effect) and ofthose 8 grew more rapidly than the rest as indicated by significantplaque numbers or the size of plaques after 7 days of incubation.Rapidly growing clones were selected for virus production in 400 mL 293F(ThermoFisher cat. No. A14528) suspension cultures. Infectious units(IU) titers were determined for the 8 clones. As shown in FIG. 25 , allselected clones demonstrated IU titers at or above the unpurified pooledvirus reference. Clones 1, 24, and 60 demonstrated at least a 9-foldincrease in IU titers relative to the unpurified pooled virus reference.

Clones 1, 24, and 60 (the 3 most productive clones) were furtheranalyzed by NGS and indicated each contained deletions in the E4 region.Two of the clones (Clone 1A & clone 24) shared an identical 727 bpmutation between E4orf2-E4orf4 (FIG. 26 ), specifically between 34,916to 35,642 bp of the wild-type ChAdV68 virus (SEQ ID NO: 1). Clone 60 wasdeleted in the E4orf1-E4orf3 region (34,980-36,516), but the deletionwas larger (1539 bp). Based on these deletions Orf 2 & 3 deletions(34,979-35,642) are common to both clone sets suggesting the Orf 2 & 3deletions contribute to the productivity improvement.

Three E4 deleted viral vectors were generated deleting the E4 regionportion deleted in Clones 1A and 24 and compared with their originalnon-E4 deleted vectors. The vectors chosen were 1) ChAdV-Empty (“Empty”)with no cassette or regulatory regions (promoter or poly-A) 2)ChAdV68.5WTnt.GFP (SEQ ID NO: 13; “GFP”), and 3) ChAdV68.5WTnt.MAG25mer(SEQ ID NO:2; “MAG”). They are all based on sequence AC_000011.1 with E1(nt577 to 3403) and E3 deleted (nt 27,125-31,825) [SEQ ID NO: 1]. Thesewere compared to the same vectors but deleted in the E4 region that weidentified (34,916 to 35,642 of SEQ ID NO:1); ChAdV68-Empty-E4deleted(SEQ ID NO: 59; “E4 Delta”), ChAdV68-GFP-E4deleted (SEQ ID NO: 58; “GFPE4 Delta”), and ChAdV68-MAG-E4deleted (SEQ ID NO: 57; “MAG E4 Delta” and“ChAdV68-MAG-E4”), respectively. These six vectors were made and viralparticle (VP) and infectious unit (IU) titers determined. Productivitywas evaluated at the 400 mL production scale. As shown in FIG. 27 , ineach comparison the E4 deleted version demonstrated increased viralparticle titers (left panel) and infectious unit titers (right panel).

Expression of the MAG cassette was compared between E4 deleted andnon-deleted vectors. As shown in FIG. 28 , Western analysis on HEK293Fcell lysates infected with ChAdV68.5WTnt.MAG25mer (“MAG”) andChAdV68-MAG-E4deleted (“MAG-E4”) viruses indicated that the E4 deletedvirus had higher levels of the MAG cassette expressed compared to thenon E4-deleted viruses.

Mice were then immunized comparing the ChAdV68.5WTnt.MAG25mer(“ChAdV68-MAG”) and its E4-deleted counterpart ChAdV68-MAG-E4deleted(“ChAdV68-E4delta”). T cell responses were analyzed for IFN-gammaproduction by ICS following stimulation with an AH1 peptide. As shown inFIG. 42A and Table 41A, immunization with the E4-deleted vectordemonstrated at least equivalent immune responses at both doses tested(1×10⁹ left panel, 1×10¹⁰ right panel), with a positive trend towards anincreased response in E4-deleted vectors.

TABLE 41A IFN-gamma production by E4 deleted ChAdV68 (ICS) AverageIFNg + Standard Treatment Dose as % of CD8 deviation N ChAdV68-MAG1.00E+10 1.040875 0.211938 8 ChAdV68-E4delta 1.00E+10 1.084125 0.2131098 ChAdV68-MAG 1.00E+09 0.61575 0.202046 8 ChAdV68-E4delta 1.00E+090.800125 0.189558 8

Rhesus macaques were then immunized with ChAdV68.5WTnt.MAG25mer(“ChAdV68-CMV-MAG”; SEQ ID NO:2) or ChAdV68-E4d-CMT-MAG (SEQ ID NO:71),with each group also administered an anti-CTLA4 antibody (Ipilimumab). Tcell responses were analyzed for IFN-gamma production by ELISpotfollowing stimulation with 6 different rhesus macaque Mamu-A*01 class Iepitopes. As shown in FIG. 42B and FIG. 42C, and quantified in Table 41B(ChAdV68-CMV-MAG) and Table 41C (ChAdV68-E4d-CMT-MAG), immunization withthe E4-deleted vector demonstrated at least equivalent immune responses,with a positive trend towards an increased response in E4-deletedvectors.

TABLE 41B Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for ChAdV68-CMV-MAG Antigen Wk Env CL9 Env TL9 Gag CM9 Gag LW9 PolSV9 Tat TL8 1 531 +/− 131 950 +/− 215 654 +/− 216 14 +/− 6 12 +/− 0 1460 +/− 272 2 399 +/− 74  887 +/− 159 924 +/− 351  0 +/− 0 0 +/− 0 1986+/− 434 3 312 +/− 101 616 +/− 155 675 +/− 212  0 +/− 0 0 +/− 0 1795 +/−481 4 533 +/− 151 851 +/− 129 1011 +/− 207  10 +/− 7 73 +/− 12 2290 +/−729

TABLE 41C Mean spot forming cells (SFC) per 10⁶ PBMCs for each epitope ±SEM for ChAdV68-E4d-CMT-MAG Antigen Wk Env CL9 Env TL9 Gag CM9 Gag LW9Pol SV9 Tat TL8 1 1037 +/− 285  966 +/− 287 1341 +/− 470 20 +/− 13 10+/− 9   2777 +/− 1180 2  707 +/− 376  905 +/− 343 1217 +/− 543 0 +/− 0 0+/− 0 1805 +/− 681 3  612 +/− 302 1038 +/− 361 1040 +/− 474 0 +/− 0 0+/− 0 1906 +/− 462 4 1237 +/− 722 1282 +/− 665 1487 +/− 760 3 +/− 2 183+/− 122 2084 +/− 943

XXI. Construction of a TETr-Regulated Cassette Expression System

A TETr-regulated viral expression system was established to minimizetranscription of nucleic acids encoded in a cassette, such as an antigenencoding cassette in a vaccine, during viral production. FIG. 43illustrates the general strategy for one example of atetracycline-controlled viral production system using the example ofantigen encoding vaccine, namely:

-   -   293F cells expressing a TET repressor protein (TETr) repress        expression of the vaccine cassette by binding to the TET        operator sequence upstream of a minimal CMV promoter    -   Transcription of the cassette sequence facilitates Adenovirus        production without the influence of cassette expression    -   Once administered in vivo, no repressor is present, and        transcription of the cassette can proceed unimpeded

FIG. 44A presents a schematic showing arrangement of one example of aTET response region, referred to as a “TETo” response region, inreference to the promoter and cassette to be expressed. The TET responseregion consists of seven repeats of the 19 bp TET operator (TETo)sequence (TCCCTATCAGTGATAGAGA; SEQ ID NO:60) linked with spacers(aaagtgaaagtcgagtttaccac; SEQ ID NO:70) between each TETo. The TETresponse region is upstream (5′) of the minimal CMV promoter (67 bp; seeSEQ ID NO:61) and the start of the cassette location. The arrangement ofthe TETo response region and promoter sequences are shown and describedin SEQ ID NO:61.

FIG. 44B presents a schematic showing arrangement of another example ofa TET response region, referred to as a “CMT” response region, inreference to the promoter and cassette to be expressed. The TET responseregion includes two repeats of the 19 bp TET operator (TETo) sequence(TCCCTATCAGTGATAGAGA; SEQ ID NO:60) linked together with a twonucleotide spacer. The TET response region is downstream (3′) of afull-length CMV promoter (605 bp; see SEQ ID NO: 64) and upstream (5′)of the start of the cassette location. The arrangement of the CMTresponse region and promoter sequences are shown and described in SEQ IDNO:64.

The TETo response region was inserted between the I-SceI and AsisI sitesof ChAdV68.5WTnt.GFP (SEQ ID NO: 13) to generate ChAdV68-TETo-GFP. ATETr sequence (tTS; SEQ ID NO: 62) was cloned into a Lentivirus pLXvector to generate pLXCMV-tTS-iPuro and used to transduce 293F cells.Sequences used in constructing the system are presented below. A clonal293F TETr line was generated after Puromycin selection. GFP transgeneexpression was evaluated to assess expression regulation by the TETrline in vitro. As shown in FIG. 45A, following infection withChAdV68-TETo-GFP virus, GFP was significantly reduced in 293F cellsexpressing the TETr (Clone 17, right panel) relative to the parental293F cell line (left panel).

A secreted embryonic alkaline phosphatase SEAP reporter construct wasgenerated using the CMT response region inserted between the I-SceI andAsisI sites of ChAdV68-Empty-E4deleted (SEQ ID NO:59) and with SEAPinserted in place of the deleted E1 (“ChAdV68-E4d-CMT-SEAP”). 293F cellswere infected at an MOI of 0.3 and 24 h later media was harvested forthe SEAP assay (Phospha-Light™ System (Applied Biosystems) using achemiluminescent substrate for the detection of secreted alkalinephosphatase) that was followed according to the manufacturersdescription. As shown in FIG. 45B, following infection withChAdV68-E4d-CMT-SEAP virus, SEAP secretion was reduced 120-fold tobackground level in 293F cells expressing the TETr (“tTS Clone 17”),with background set using a ChAdV68 vector expressing a controlexpression cassette, relative to the parental 293F cell line (“293F”).Thus, adenoviral cassettes expressed from a TETr-controlled promoterdemonstrate reduced cassette expression when used in TETr-expressingcell lines in vitro.

TETo response region between I-SceI and AsisI sites of ChAdV68 vector backbone.One of seven repeats of the 19 bp TETo sequences is bold italicized.The minimal CMV promoter is bold (SEQ ID NO: 61)ccatgttgacattgattattgactagttattaaagtact tccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgatattaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgaccactccctatcagtgatagagaaaagtgaaagtcgagacggtacccggctcgaggtaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggag TETr sequence (tTS) nucleic acid sequence  (SEQ ID NO: 62)ATGAGCAGACTGGACAAGAGCAAAGTGATCAACAGCGCCCTGGAACTGCTGAACGAAGTGGGCATCGAGGGCCTGACAACCAGAAAGCTGGCCCAGAAGCTGGGCGTTGAGCAGCCTACACTGTATTGGCACGTGCGGAACAAGCAGACCCTGATGAATATGCTGAGCGAGGCCATCCTGGCCAAGCACCATACAAGATCTGCCCCTCTGCCAACCGAGAGCTGGCAGCAGTTTCTGCAAGAGAACGCCCTGAGCTTCAGAAAGGCCCTGCTGGTGCATAGAGATGGCGCCAGACTGCACATCGGCACATCTCCCACACCTCCACAGTTTGAGCAGGCTGAGGCACAGCTGAGATGTCTGTGTGATGCCGGCTTTAGCGTGGAAGAGGCCCTGTTCATCCTGCAGTCCATCAGCCACTTTACACTGGGCGCCGTGCTGGAAGAACAGGCCACCAACCAGATCGAGAACAACCACGTGATCGACGCTGCCCCTCCACTGCTGCAAGAGGCCTTCAATATCCAAGCCAGAACCAGCGCCGAGATGGCCTTCCACTTTGGCCTGAAGTCCCTGATCTTTGGCTTCAGCGCCCAGCTGGACGAGAAGAAGCACACACCTATCGAGGACGGCAACAAGCCCAAGAAGAAGCGGAAGCTGGCCGTCAGCGTGACCTTTGAAGATGTGGCCGTGCTGTTCACCCGGGACGAGTGGAAGAAACTGGACCTGAGCCAGCGGAGCCTGTACCGGGAAGTGATGCTGGAAAACTACAGCAACCTGGCCTCCATGGCCGGCTTTCTGTTCACCAAGCCTAAAGTGATCTCCCTGCTTCAGCAGGGCGAAGATCCTTGGTAA TETr sequence (tTs) amino acid sequetic  (SEQ ID NO: 63)MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVRNKQTLMNMLSEAILAKHHTRSAPLPTESWQQFLQENALSFRKALLVHRDGARLHIGTSPTPPQFEQAEAQLRCLCDAGFSVEEALFILQSISHFTLGAVLEEQATNQIENNHVIDAAPPLLQEAFNIQARTSAEMAFHFGLKSLIFGFSAQLDEKKHTPIEDGNKPKKKRKLAVSVTEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASMAGFLFTKPKVISLLQQGEDPWCMT response region between I-SceI and AsisI sites of ChAdV68 vector backbone.The two repeats of the 19 bp TETo sequences are bolded.The full-length CMV promoter is italicized. (SEQ ID NO: 64)GACATTGATTATTGACTAGTTGTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGCTTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAAGTACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCACTTACATCTACGTATTAGTCATCGCTAATTACCATCTGTGTGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATC

XXII. Viral Production in the TETr-Regulated Cassette Expression System

The TETo response region was inserted between the I-SceI and AsisI sitesof ChAdV68.5WTnt.MAG25mer (“ChAdV68-CMV-MAG”; SEQ ID NO:2) to generate aChAdV68-TETo-MAG virus (SEQ ID NO:65) expressing a model antigencassette under control of a TET regulated promoter. Viral production wascompared between cell lines expressing TETr (Clone 17) and the parentalcell line that did not express TETr (293F). As shown in FIG. 46 , viralproduction was improved as assessed by viral particle (VP; top panels)and infectious unit (IU; bottom panels) titers across three independentreplicates.

Viral production of a ChAdV68-TETo-MAG virus produced in a cell lineexpressing TETr (Clone 17) was also compared to a virus lacking the TETosequences (“ChAdV68-CMV-MAG”). As shown in FIG. 47A, viral productionwas improved by 3.4-fold for the ChAdV68-TETo-MAG virus relative toChAdV68-CMV-MAG. These results indicate reduction in in vitro expressionof the delivered cassette transgenes translated into more consistent andimproved virus productivity.

Viral production produced in a cell line expressing TETr (tTS Clone 17)was further compared for a series of viral constructs, includingconstructs featuring E4 deletions and TET response elements. Theconstructs all expressed the same, control tumor-specific neoantigen(TSNA) cassette. The general backbone featuring E1/E3 deletions and 5nucleotide substitutions was the same as ChAdV68.5WTnt.MAG25mer (SEQ IDNO:2), with TETo and CMT response regions inserted between the I-SceIand AsisI sites, as indicated, and the MAG25mer cassette substituted forthe TSNA cassette. The deleted E4 region was that identified above(deletion 34,916 to 35,642 of SEQ ID NO:1). The various constructsexamined are described below:

-   -   ChAdV68-CMV-TSNA; E1/E3 deleted, full-length CMV promoter    -   ChAdV68-CT-TSNA; E1/E3 deleted, full-length CMV promoter, TSNA        cassette codon optimized using an alternate codon optimization        (SEQ ID NO:66)    -   ChAdV68-TETo-TSNA; E1/E3 deleted, 7 repeats of TETo linked with        spacers upstream (5′) of minimal CMV promoter (“TETo” response        region) (SEQ ID NO:67)    -   ChAdV68-CMT-TSNA; E1/E3 deleted, 2 repeats of TETo directly        linked together downstream (3′) of full-length CMV promoter        (“CMT” response region) (SEQ ID NO:68)    -   ChAdV68-E4d-CMT-TSNA; E1/E3/E4 deleted, 2 repeats of TETo        directly linked together downstream (3′) of full-length CMV        promoter (SEQ ID NO:69)

As shown in FIG. 47B, viral production for the ChAdV68-CT-TSNA,ChAdV68-TETo-TSNA, ChAdV68-CMT-TSNA, and ChAdV68-E4d-CMT-TSNA viruseswas improved by about 6-fold, 39-fold, 137-fold, or 300-fold relative toChAdV68-CMV-TSNA. respectively. The ratio of viral particles toinfectious units was also assessed to measure the virus's infectiouscapability and is calculated by dividing the virus particle (VP)titer/mL by the infectious unit (IU) titer/mL, where a lower ratiorepresents a higher infectivity per particle (a ratio of 1:1 representsa perfect ratio of every particle being infectious). As shown in Table42A, TET-controlled vectors ChAdV68-TETo-TSNA, ChAdV68-CMT-TSNA, andChAdV68-E4d-CMT-TSNA all demonstrated improved infectious capabilityrelative to ChAdV68-CMV-TSNA, with CMT vectors demonstrating the bestinfectious capability.

Viral production of another series of viral constructs, includingconstructs featuring E4 deletions and TET response elements, wasassessed for constructs featuring either a large model antigen cassette(50XXL; see FIG. 29 and Tables 32-34) or M2.2 model antigen cassette.The general backbone featuring E1/E3 deletions and 5 nucleotidesubstitutions was the same as ChAdV68.5WTnt.MAG25mer (SEQ ID NO:2), withTETo and CMT response regions inserted between the I-SceI and AsisIsites, as indicated, and the MAG25mer cassette substituted for theindicated cassettes. The deleted E4 region was that identified above(deletion 34,916 to 35,642 of SEQ ID NO:1). The various constructsexamined are described below:

-   -   ChAdV68-CMV-50 XXL; E1/E3 deleted, full length CMV promoter,        50XXL cassette codon optimized using Genscript codon        optimization tool    -   ChAdV68-CMT-50XXL; E1/E3 deleted, 2 repeats of TETo directly        linked together downstream (3′) of full-length CMV promoter,        50XXL cassette codon optimized using Genscript codon        optimization tool    -   ChAdV68-CT-50XXL; E1/E3 deleted, full length CMV promoter, 50XXL        cassette codon optimized using an alternate codon optimization        tool    -   ChAdV68-E4d-CMT-50XXL; E1/E3/E4 deleted, 2 repeats of TETo        directly linked together downstream (3′) of full-length CMV        promoter; 50XXL cassette codon optimized using Genscript codon        optimization tool    -   ChAdV68-CMV-M2.2; E1/E3 deleted, full length CMV promoter, M2.2        cassette codon optimized using Genscript codon optimization tool    -   ChAdV68-CMT-M2.2; E1/E3 deleted, 2 repeats of TETo directly        linked together downstream (3′) of full-length CMV promoter,        M2.2 cassette codon optimized using Genscript codon optimization        tool    -   ChAdV68-E4d-CMT-M2.2; E1/E3/E4 deleted, 2 repeats of TETo        directly linked together downstream (3′) of full-length CMV        promoter, M2.2 cassette codon optimized using Genscript codon        optimization tool

As shown in FIG. 47C, viral production for model antigen cassettes 50XXLand M2.2 was improved by the use adenoviral vectors having a CMTresponse region in a tTS expressing cell line. For example, viralproduction was almost 10-fold greater for ChAdV68-CMT-50XXL in the tTSexpressing cell line (left panel; middle column) relative to a parental293F cell line (left panel; second column from left), and 15-foldgreater for ChAdV68-CMT-M2.2 (right panel; middle column) relative to avector lacking the CMT response region in a parental 293F cell line(right panel; left column). In the case of 50XXL constructs, furtherimprovements in viral production were achieved by combining a CMTresponse region with an E4 deletion (left panel middle column vs leftpanel right column). Improvements were also achieved under certaincircumstances by alternative codon optimization (as shown forChAdV68-CT-50XXL). The ratio of viral particles to infectious units wasalso assessed. As shown in Table 42C, TET-controlled vectors in a E4deleted background all demonstrated improved infectious capabilityrelative to vectors without an E4 deletion and TET response element.

TABLE 42A Viral particle to infectious unit ratio TSNA constructsConstruct VP:IU Ratio ChAdV68-CMV-TSNA 591:1  ChAdV68-CT-TSNA 63:1ChAdV68-TETo-TSNA 135:1  ChAdV68-CMT-TSNA 22:1 ChAdV68-E4d-CMT-TSNA 34:1

TABLE 42B Viral particle to infectious unit ratio 50XXL constructsConstruct VP:IU Ratio ChAdV68-CMV-50XXL 260:1  ChAdV68-E4d-CMT-50XXL32:1 ChAdV68-CMV-M2.2 662:1  ChAdV68-E4d-CMT-M2.2 50:1

ChAdV68-TETo-MAG (SEQ ID NO: 65)CATCATCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATCCATGTTGACATTGATTATTGACTAGTTATTAAAGTACTTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGCCACCATGGCCGGGATGTTCCAGGCACTGTCCGAAGGCTGCACACCCTATGATATTAACCAGATGCTGAATGTCCTGGGAGACCACCAGGTCTCTGGCCTGGAGCAGCTGGAGAGCATCATCAACTTCGAGAAGCTGACCGAGTGGACAAGCTCCAATGTGATGCCTATCCTGTCCCCACTGACCAAGGGCATCCTGGGCTTCGTGTTTACCCTGACAGTGCCTTCTGAGCGGGGCCTGTCTTGCATCAGCGAGGCAGACGCAACCACACCAGAGTCCGCCAATCTGGGCGAGGAGATCCTGTCTCAGCTGTACCTGTGGCCCCGGGTGACATATCACTCCCCTTCTTACGCCTATCACCAGTTCGAGCGGAGAGCCAAGTACAAGAGACACTTCCCAGGCTTTGGCCAGTCTCTGCTGTTCGGCTACCCCGTGTACGTGTTCGGCGATTGCGTGCAGGGCGACTGGGATGCCATCCGGTTTAGATACTGCGCACCACCTGGATATGCACTGCTGAGGTGTAACGACACCAATTATTCCGCCCTGCTGGCAGTGGGCGCCCTGGAGGGCCCTCGCAATCAGGATTGGCTGGGCGTGCCAAGGCAGCTGGTGACACGCATGCAGGCCATCCAGAACGCAGGCCTGTGCACCCTGGTGGCAATGCTGGAGGAGACAATCTTCTGGCTGCAGGCCTTTCTGATGGCCCTGACCGACAGCGGCCCCAAGACAAACATCATCGTGGATTCCCAGTACGTGATGGGCATCTCCAAGCCTTCTTTCCAGGAGTTTGTGGACTGGGAGAACGTGAGCCCAGAGCTGAATTCCACCGATCAGCCATTCTGGCAGGCAGGAATCCTGGCAAGGAACCTGGTGCCTATGGTGGCCACAGTGCAGGGCCAGAATCTGAAGTACCAGGGCCAGAGCCTGGTCATCAGCGCCTCCATCATCGTGTTTAACCTGCTGGAGCTGGAGGGCGACTATCGGGACGATGGCAACGTGTGGGTGCACACCCCACTGAGCCCCAGAACACTGAACGCCTGGGTGAAGGCCGTGGAGGAGAAGAAGGGCATCCCAGTGCACCTGGAGCTGGCCTCCATGACCAATATGGAGCTGATGTCTAGCATCGTGCACCAGCAGGTGAGGACATACGGACCCGTGTTCATGTGCCTGGGAGGCCTGCTGACCATGGTGGCAGGAGCCGTGTGGCTGACAGTGCGGGTGCTGGAGCTGTTCAGAGCCGCCCAGCTGGCCAACGATGTGGTGCTGCAGATCATGGAGCTGTGCGGAGCAGCCTTTCGCCAGGTGTGCCACACCACAGTGCCATGGCCCAATGCCTCCCTGACCCCCAAGTGGAACAATGAGACAACACAGCCTCAGATCGCCAACTGTAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTATAGCGTGAGGGATACCCTGTGGCCCCGCGTGACATACCACATGAATAAGTACGCCTATCACATGCTGGAGAGGCGCGCCAAGTATAAGAGAGGCCCTGGCCCAGGCGCAAAGTTTGTGGCAGCATGGACCCTGAAGGCCGCCGCCGGCCCCGGCCCCGGCCAGTATATCAAGGCTAACAGTAAGTTCATTGGAATCACAGAGCTGGGACCCGGACCTGGATAATGAGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTACCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGFCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACCGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGTACTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCGGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCCGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCTGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCAATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGCAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGGGGGAGGGAAGAACAGGAAGAACCATGATTAACTTTTAATCCAAACGGTCTCGGAGTACTTCAAAATGAAGATCGCGGAGATGGCACCTCTCGCCCCCGCTGTGTTGGTGGAAAATAACAGCCAGGTCAAAGGTGATACGGTTCTCGAGATGTTCCACGGTGGCTTCCAGCAAAGCCTCCACGCGCACATCCAGAAACAAGACAATAGCGAAAGCGGGAGGGTTCTCTAATTCCTCAATCATCATGTTACACTCCTGCACCATCCCCAGATAATTTTCATTTTTCCAGCCTTGAATGATTCGAACTAGTTCCTGAGGTAAATCCAAGCCAGCCATGATAAAGAGCTCGCGCAGAGCGCCCTCCACCGGCATTCTTAAGCACACCCTCATAATTCCAAGATATTCTGCTCCTGGTTCACCTGCAGCAGATTGACAAGCGGAATATCAAAATCTCTGCCGCGATCCCTGAGCTCCTCCCTCAGCAATAACTGTAAGTACTCTTTCATATCCTCTCCGAAATTTTTAGCCATAGGACCACCAGGAATAAGATTAGGGCAAGCCACAGTACAGATAAACCGAAGTCCTCCCCAGTGAGCATTGCCAAATGCAAGACTGCTATAAGCATGCTGGCTAGACCCGGTGATATCTTCCAGATAACTGGACAGAAAATCGCCCAGGCAATTTTTAAGAAAATCAACAAAAGAAAAATCCTCCAGGTGGACGTTTAGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATGGChAdV68-CT-TSNA (SEQ ID NO: 66)CATCATCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATGACATTGATTATTGACTAGTTGTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGCCACCATGGCTGGCATGACCGAGTACAAGCTGGTGGTAGTGGGAGCAGGGGATGTTGGAAAATCAGCCTTGACTATTCAGCTCATCCAGGGCACAGATCTGGATCACCAGGAGAAATGTCTCTCGCGACTGTACGACCACATGCCTGAGGGTCTGACCCCCCTTATGGGAGTGTCGTCCTCTTCTGCGCTGGCCCGTCTCGGATTACCCATGGATAAACTCAATAAAATCACCGCCCCGGCGAGCCAGAAGTTAAGACAACTGCAAAAGATGGAAACTCCTGAACTACTGCCCTGTGGGTATCTTGTAGAAGAAAATACCACGATCTCTGTGACAGTGAAGGGCCTGGAGGCTCAGAATAAGATCAAAGGGTGCACTGGGTCGGTGAACATGACTTTACAGAGAGCCAGCGCAGCTCCTAAGACTGGTGGCGGGGGTGAAGCCGCTGCATACAACAACACTCTTGTGGCACGGCACGTGCCCCAGATACCAAAGCCCGATTCCTTGGTGGGGCTTAGTGATGAGTTGGGGAAGCGGGACACTTTTGCAGAGTCTCTGATTCGTAGGATGGCATCCGCGGGCTACCTGTTCCTGGACATCATCACATACGTGGTCTTTGCTGTAACCTTCGTGCTTGGTGTTTTAGGAGGGCTGAACACAGAAACCAATGAGAAGGCTTTAGAAGCTGTGTTTGGCAAGTATGGAAGAATAGTGGAGGTGCTGGGGGGCCGGTCATGCGAGGAGCTGACGGCGGTACTTCCTCCACCTCAGCTTTTGGGCAGGAGATTTAACTTCTTCTCATACTCCTATGTGGCCGCAGGAAGTTCCGGGAATAACTATGACCTCATGGCCCAACCCATCACGCCCGGGCCCGACACAACCCCGTTACCAGTGACCGATACTAGTTCCGTGAGTACAGGCCACGCCACCAGCCTGCCTGTGACTGACGCTGGACTCAGGGTTACAGAGAGTAAGGGGCACAGCGATTCATGGCACCTGTCTTTGGATACGGCCATCAGGGTCAACACCCCTAAACTGGTGTCCGAGGTTGAGGAACTCAACAAAAGCATTACAGCGCTACGAGAAAAGCTACTGCAGATGGTGGAGGCCGACAGACCCGGAAACCTCTTCATTGGGGGCTTAAATACAGAGACTAATGAAGACAGCCCGGTCAAGGATGAAGTAGTGGTGAATGATCAGTGGGGACAGAACTGCAGCTGCCACCACGGCGGTTACGAGTTTCCGGACCTGCACCGCACCATCGTGTCTGAGTGTGACGTGTACCTCACCTACATGCTGCGCCAGGCCGCCCTTCAGCTGTTCTTTGATCTCTACCACTCCATTCCGTCAAGCTTCAGCCCCTTAGTCCTCAGCTGTTTAGTGCAGCCCTTGGAAGATGTGGAGGTCATGGAGAAGGACGGCACCACATTCTCCTGTGAAGTTTCTCATGACGAGGTTCCTCGGACATATGGACCCGTGTTTATGTGTCTGGGAGGACTGCTGACCATGGTGGCTGGAGCTGTTTGGCTGACAGTTGGACCCGGACCAGGCGCCAAATTTGTTGCTGCTTGGACACTGAAAGCTGCTGCTGGGCCCGGACCAGGCCAGTACATCAAGGCCAACTCTAAGTTTATCGGCATCACCGAATTGGGACCTGGACCCGGCTAGTAGTGAGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTACCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGACGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCGGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCCGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCTGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCAATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGC CTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGCTCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGGTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGACGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTACCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGOTGCCGCCCACGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACCGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCACGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGGGGGAGGGAAGAACAGGAAGAACCATGATTAACTTTTAATCCAAACGGTCTCGGAGTACTTCAAAATGAAGATCGCGGAGATGGCACCTCTCGCCCCCGCTGTGTTGGTGGAAAATAACAGCCAGGTCAAAGGTGATACGGTTCTCGAGATGTTCCACGGTGGCTTCCAGCAAAGCCTCCACGCGCACATCCAGAAACAAGACAATAGCGAAAGCGGGAGGGTTCTCTAATTCCTCAATCATCATGTTACACTCCTGCACCATCCCCAGATAATTTTCATTTTTCCAGCCTTGAATGATTCGAACTAGTTCCTGAGGTAAATCCAAGCCAGCCATGATAAAGAGCTCGCGCAGAGCGCCCTCCACCGGCATTCTTAAGCACACCCTCATAATTCCAAGATATTCTGCTCCTGGTTCACCTGCAGCAGATTGACAAGCGGAATATCAAAATCTCTGCCGCGATCCCTGAGCTCCTCCCTCAGCAATAACTGTAAGTACTCTTTCATATCCTCTCCGAAATTTTTAGCCATAGGACCACCAGGAATAAGATTAGGGCAAGCCACAGTACAGATAAACCGAAGTCCTCCCCAGTGAGCATTGCCAAATGCAAGACTGCTATAAGCATGCTGGCTAGACCCGGTGATATCTTCCAGATAACTGGACAGAAAATCGCCCAGGCAATTTTTAAGAAAATCAACAAAAGAAAAATCCTCCAGGTGGACGTTTAGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCCACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATG ChAdV68-TETo-TSNA (SEQ ID NO: 67)CCATCTTCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATCCATGTTGACATTGATTATTGACTAGTTATTAAAGTACTTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGCCACCATGGCTGGCATGACCGAGTACAAGCTAGTCGTTGTGGGAGCTGGAGATGTGGGCAAATCTGCTCTGACCATTCAGCTGATTCAGGGCACAGATCTGGATCACCAGGAGAAGTGTCTGAGCAGGCTGTACGACCACATGCCAGAAGGATTAACCCCTCTTATGGGAGTGAGCTCTTCTTCTGCTCTGGCCAGACTGGGACTGCCTATGGATAAGCTGAACAAGATCACAGCTCCTGCCTCTCAGAAACTGAGACAGCTGCAGAAGATGGAGACCCCTGAACTGCTGCCTTGTGGATATCTGGTGGAGGAGAATACCACAATCAGCGTGACCGTGAAAGGCCTGGAAGCCCAGAACAAGATCAAAGGCTGTACCGGCTCTGTGAATATGACACTGCAGAGAGCTTCTGCCGCCCCTAAGACAGGAGGAGGAGGAGAAGCTGCTGCCTACAATAATACATTAGTGGCCAGACATGTGCCCCAGATCCCTAAGCCTGACAGCCTGGTTGGCCTGAGCGATGAATTAGGAAAAAGAGACACATTTGCCGAGAGCCTGATCCGGAGAATGGCCTCTGCCGGCTACCTGTTCCTGGATATCATCACATATGTTGTGTTTGCCGTGACCTTCGTGCTGGGAGTTCTGGGCGGCCTGAATACCGAGACCAATGAAAAAGCTCTTGAAGCCGTGTTTGGCAAGTACGGCAGAATCGTGGAGGTGCTGGGCGGCAGATCTTGTGAAGAATTAACAGCTGTGTTACCACCTCCTCAGCTGCTTGGCAGACGGTTCAACTTCTTCAGCTACAGCTACGTTGCTGCTGGCTCTTCTGGCAACAACTACGACCTGATGGCCCAGCCTATTACACCTGGACCTGATACAACACCTCTGCCTGTGACCGATACATCTTCTGTGTCTACCGGACACGCCACATCTCTGCCAGTGACAGATGCTGGACTGAGAGTGACAGAGTCTAAAGGACACAGCGATTCTTGGCACCTGAGCCTGGATACAGCCATCAGGGTGAATACCCCTAAGCTGGTTTCTGAAGTGGAAGAGCTGAACAAGAGCATCACCGCCCTGAGGGAGAAGTTACTGCAGATGGTGGAAGCCGATAGACCTGGAAACCTGTTTATTGGAGGCCTGAACACCGAGACCAATGAGGACTCTCCCGTGAAGGATGAAGTGGTGGTGAACGATCAATGGGGCCAGAATTGTAGCTGCCATCATGGAGGCTACGAGTTCCCTGATCTGCACAGGACAATCGTGTCTGAGTGCGATGTGTATCTGACCTACATGCTGAGACAGGCTGCTCTGCAGCTGTTCTTCGACCTGTATCACAGCATCCCTAGCAGCTTTTCTCCTCTGGTTCTGAGCTGTCTGGTGCAGCCTCTGGAAGATGTGGAAGTGATGGAGAAGGATGGCACAACCTTTAGCTGTGAGGTGAGCCACGATGAGGTGCCTCGGACATATGGACCCGTGTTTATGTGTCTGGGAGGACTGCTGACCATGGTGGCTGGAGCTGTTTGGCTGACAGTTGGACCCGGACCAGGCGCCAAATTTGTTGCTGCTTGGACACTGAAAGCTGCTGCTGGGCCCGGACCAGGCCAGTACATCAAGGCCAACTCTAAGTTTATCGGCATCACCGAATTGGGACCTGGACCCGGCTAGTAGTGAGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTACCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGGTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCGGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCCGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCTGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCAAATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACOCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGGGGGAGGGAAGAACAGGAAGAACCATGATTAACTTTTAATCCAAACGGTCTCGGAGTACTTCAAAATGAAGATCGCGGAGATGGCACCTCTCGCCCCCGCTGTGTTGGTGGAAAATAACAGCCAGGTCAAAGGTGATACGGTTCTCGAGATGTTCCACGGTGGCTTCCAGCAAAGCCTCCACGCGCACATCCAGAAACAAGACAATAGCGAAAGCGGGAGGGTTCTCTAATTCCTCAATCATCATGTTACACTCCTGCACCATCCCCAGATAATTTTCATTTTTCCAGCCTTGAATGATTCGAACTAGTTCCTGAGGTAAATCCAAGCCAGCCATGATAAAGAGCTCGCGCAGAGCGCCCTCCACCGGCATTCTTAAGCACACCCTCATAATTCCAAGATATTCTGCTCCTGGTTCACCTGCAGCAGATTGACAAGCGGAATATCAAAATCTCTGCCGCGATCCCTGAGCTCCTCCCTCAGCAATAACTGTAAGTACTCTTTCATATCCTCTCCGAAATTTTAGCCATAGGACCACCAGGAATAAGATTAGGGCAAGCCACAGTACAGATAAACCGAAGTCCTCCCCAGTGAGCATTGCCAAATGCAAGACTGCTATAAGCATGCTGGCTAGACCCGGTGATATCTTCCAGATAACTGGACAGAAAATCGCCCAGGCAATTTTTAAGAAAATCAACAAAAGAAAAATCCTCCAGGTGGACGTTTAGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATGG ChAdV68-CMT-TSNA (SEQ ID NO: 68)CATCATCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATGACATTGATTATTGACTAGTTGTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGCCACCATGGCTGGCATGACCGAGTACAAGCTAGTCGTTGTGGGAGCTGGAGATGTGGGCAAATCTGCTCTGACCATTCAGCTGATTCAGGGCACAGATCTGGATCACCAGGAGAAGTGTCTGAGCAGGCTGTACGACCACATGCCAGAAGGATTAACCCCTCTTATGGGAGTGAGCTCTTCTTCTGCTCTGGCCAGACTGGGACTGCCTATGGATAAGCTGAACAAGATCACAGCTCCTGCCTCTCAGAAACTGAGACAGTGCAGAAGATGGAGACCCCTGAACTGCTGCCTTGTGGATATCTGGTGGAGGAGAATACCACAATCAGCGTGACCGTGAAAGGCCTGGAAGCCCAGAACAAGATCAAAGGCTGTACCGGCTCTGTGAATATGACACTGCAGAGAGCTTCTGCCGCCCCTAAGACAGGAGGAGGAGGAGAAGCTGCTGCCTACAATAATACATTAGTGGCCAGACATGTGCCCCAGATCCCTAAGCCTGACAGCCTGGTTGGCCTGAGCGATGAATTAGGAAAAAGAGACACATTTGCCGAGAGCCTGATCCGGAGAATGGCCTCTGCCGGCTACCTGTTCCTGGATATCATCACATATGTTGTGTTTGCCGTGACCTTCGTGCTGGGAGTTCTGGGCGGCCTGAATACCGAGACCAATGAAAAAGCTCTTGAAGCCGTGTTTGGCAAGTACGGCAGAATCGTGGAGGTGCTGGGCGGCAGATCTTGTGAAGAATTAACAGCTGTGTTACCACCTCCTCAGCTGCTTGGCAGACGGTTCAACTTCTTCAGCTACAGCTACGTTGCTGCTGGCTCTTCTGGCAACAACTACGACCTGATGGCCCAGCCTATTACACCTGGACCTGATACAACACCTCTGCCTGTGACCGATACATCTTCTGTGTCTACCGGACACGCCACATCTCTGCCAGTGACAGATGCTGGACTGAGAGTGACAGAGTCTAAAGGACACAGCGATTCTTGGCACCTGAGCCTGGATACAGCCATCAGGGTGAATACCCCTAAGCTGGTTTCTGAAGTGGAAGAGCTGAACAAGAGCATCACCGCCCTGAGGGAGAAGTTACTGCAGATGGTGGAAGCCGATAGACCTGGAAACCTGTTTATTGGAGGCCTGAACACCGAGACCAATGAGGACTCTCCCGTGAAGGATGAAGTGGTGGTGAACGATCAATGGGGCCAGAATTGTAGCTGCCATCATGGAGGCTACGAGTTCCCTGATCTGCACAGGACAATCGTGTCTGAGTGCGATGTGTATCTGACCTACATGCTGAGACAGGCTGCTCTGCAGCTGTTCTTCGACCTGTATCACAGCATCCCTAGCAGCTTTTCTCCTCTGGTTCTGAGCTGTCTGGTGCAGCCTCTGGAAGATGTGGAAGTGATGGAGAAGGATGGCACAACCTTTAGCTGTGAGGTGAGCCACGATGAGGTGCCTCGGACATATGGACCCGTGTTTATGTGTCTGGGAGGACTGCTGACCATGGTGGCTGGAGCTGTTTGGCTGACAGTTGGACCCGGACCAGGCGCCAAATTTGTTGCTGCTTGGACACTGAAAGCTGCTGCTGGGCCCGGACCAGGCCAGTACATCAAGGCCAACTCTAAGTTTATCGGCATCACCGAATTGGGACCTGGACCCGGCTAGTAGTGAGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTTCCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATTGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCGGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACOGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCATTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAOCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCCGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCTGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCAATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTOGAGGAGGAGCTGCCTCCCCTGGACAAGCGOGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCGTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGCCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCATATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACACGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGGGGGAGGGAAGAACAGGAAGAACCATGATTAACTTTTAATCCAAACGGTCTCGGAGTACTTCAAAATGAAGATCGCGGAGATGGCACCTCTCGCCCCCGCTGTGTTGGTGGAAAATAACAGCCAGGTCAAAGGTGATACGGTTCTCGAGATGTTCCACGGTGGCTTCCAGCAAAGCCTCCACGCGCACATCCAGAAACAAGACAATAGCGAAAGCGGGAGGGTTCTCTAATTCCTCAATCATCATGTTACACTCCTGCACCATCCCCAGATAATTTTCATTTTTCCAGCCTTGAATGATTCGAACTAGTTCCTGAGGTAAATCCAAGCCAGCCATGATAAAGAGCTCGCGCAGAGCGCCCTCCACCGGCATTCTTAAGCACACCCTCATAATTCCAAGATATTCTGCTCCTGGTTCACCTGCAGCAGATTGACAGCGGAATATCAAAATCTCTGCCGCGATCCCTGAGCTCCTCCCTCAGCAATAACTGTAAGTACTCTTTCATATCCTCTCCGAAATTTTTAGCCATAGGACCACCAGGAATAAGATTAGGGCAAGCCACAGTACAGATAAACCGAAGTCCTCCCCAGTGAGCATTGCCAAATGCAAGACTGCTATAAGCATGCTGGCTAGACCCGGTGATATCTTCCAGATAACTGGACAGAAAATCGCCCAGGCAATTTTTAAGAAAATCAACAAAAGAAAAATCCTCCAGGTGGACGTTTAGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATGChAdV68-E4d-CMT-TSNA (SEQ ID NO: 69)CATCATCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATGACATTGATTATTGACTAGTTGTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGCCACCATGGCTGGCATGACCGAGTACAAGCTAGTCGTTGTGGGAGCTGGAGATGTGGGCAAATCTGCTCTGACCATTCAGCTGATTCAGGGCACAGATCTGGATCACCAGGAGAAGTGTCTGAGCAGGCTGTACGACCACATGCCAGAAGGATTAACCCCTCTTATGGGAGTGAGCTCTTCTTCTGCTCTGGCCAGACTGGGACTGCCTATGGATAAGCTGAACAAGATCACAGCTCCTGCCTCTCAGAAACTGAGACAGCTGCAGAAGATGGAGACCCCTGAACTGCTGCCTTGTGGATATCTGGTGGAGGAGAATACCACAATCAGCGTGACCGTGAAAGGCCTGGAAGCCCAGAAACAAGATCAAAGGCTGTACCGGCTCTGTGAATATGACACTGCAGAGAGCTTCTGCCGCCCCTAAGACAGGAGGAGGAGGAGAAGCTGCTGCCTACAATAATACATTAGTGGCCAGACATGTGCCCCAGATCCCTAAGCCTGACAGCCTGGTTGGCCTGAGCGATGAATTAGGAAAAAGAGACACATTTGCCGAGAGCCTGATCCGGAGAATGGCCTCTGCCGGCTACCTGTTCCTGGATATCATCACATATGTTGTGTTTGCCGTGACCTTCGTGCTGGGAGTTCTGGGCGGCCTGAATACCGAGACCAATGAAAAAGCTCTTGAAGCCGTGTTTGGCAAGTACGGCAGAATCGTGGAGGTGCTGGGCGGCAGATCTTGTGAAGAATTAACAGCTGTGTTACCACCTCCTCAGCTGCTTGGCAGACGGTTCAACTTCTTCAGCTACAGCTACGTTGCTGCTGGCTCTTCTGGCAACAACTACGACCTGATGGCCCAGCCTATTACACCTGGACCTGATACAACACCTCTGCCTGTGACCGATACATCTTCTGTGTCTACCGGACACGCCACATCTCTGCCAGTGACAGATGCTGGACTGAGAGTGACAGAGTCTAAAGGACACAGCGATTCTTGGCACCTGAGCCTGGATACAGCCATCAGGGTGAATACCCCTAAGCTGGTTTCTGAAGTGGAAGAGCTGAACAAGAGCATCACCGCCCTGAGGGAGAAGTTACTGCAGATGGTGGAAGCCGATAGACCTGGAAACCTGTTTATTGGAGGCCTGAACACCGAGACCAATGAGGACTCTCCCGTGAAGGATGAAGTGGTGGTGAACGATCAATGOGGCCAGAATTGTAGCTGCCATCATGGAGGCTACGAGTTCCCTGATCTGCACAGGACAATCGTGTCTGAGTGCGATGTGTATCTGACCTACATGCTGAGACAGGCTGCTCTGCAGCTGTTCTTCGACCTGTATCACAGCATCCCTAGCAGCTTTTCTCCTCTGGTTCTGAGCTGTCTGGTGCAGCCTCTGGAAGATGTGGAAGTGATGGAGAAGGATGGCACAACCTTTAGCTGTGAGGTGAGCCACGATGAGGTGCCTCGGACATATGGACCCGTGTTTATGTGTCTGGGAGGACTGCTGACCATGGTGGCTGGAGCTGTTTGGCTGACAGTTGGACCCGGACCAGGCGCCAAATTTGTTGCTGCTTGGACACTGAAAGCTGCTGCTGGGCCCGGACCAGGCCAGTACATCAAGGCCAACTCTAAGTTTATCGGCATCACCGAATTGGGACCTGGACCCGGCTAGTAGTGAGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAGGTTCCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCGGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCTCTTTTGTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGACCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGCGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCCGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGCATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCTGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCAATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAGCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATXGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATG

XXIII. Immunogenicity in the TETr-Regulated Cassette Expression System

Balb/c mice were immunized with 1×10¹⁰ VP of ChAdV68 vaccines expressinga model antigen cassette either under control of normal CMV promoter(ChAdV-MAG) or a TETo regulated promoter (TET-ChAdV-MAG). 12 d postvaccination spleens were harvested and single cell suspensions made.Antigen-specific IFN-gamma production in CD8 T cells was measured usingICS. As shown in FIG. 48 and Table 43, in vivo efficacy was the same orbetter when mice were immunized with the antigen cassette expressed fromthe TETo regulated promoter. Thus, the regulated ChAd vector was equallypotent, and potentially more so, at inducing CD8+ immune responses tothe vaccine targets in vivo.

As described in greater detail above, Rhesus macaques were alsoimmunized with ChAdV68.5WTnt.MAG25mer (“ChAdV68-CMV-MAG”; SEQ ID NO:2)or ChAdV68-E4d-CMT-MAG (SEQ ID NO:71), with each group also administeredan anti-CTLA4 antibody (Ipilimumab). T cell responses were analyzed forIFN-gamma production by ELISpot following stimulation with 6 differentrhesus macaque Mamu-A*01 class I epitopes. As shown in FIG. 42B and FIG.42C, and quantified in Table 41B (ChAdV68-CMV-MAG) and Table 41C(ChAdV68-E4d-CMT-MAG), immunization with a construct featuring a “CMT”response region in E4-deleted vector background demonstrated at leastequivalent immune responses, with a positive trend towards an increasedresponse in CMT-E4-deleted vectors.

TABLE 43 % CD8+ response in ChAdV68-MAG and ChAdV68-Teto-MAG immunizedmice ChAdV68- ChAdV68- MAG1e10 Mouse % TETo- Mouse % VP # CD8+ MAG 1e10VP # CD8+ 1 9.35 1 17.58 2 9.31 2 16.88 3 17.60 3 18.93 4 10.08 4 9.59 56.06 5 24 6 8.15 6 16.28 7 10.08 7 18.92 8 9.87 8 22.24 Median 9.61Median 18.25

XXIV. Selection of Patient Populations

One or more antigens are used to formulate a vaccine composition using amodified adenovirus, such as the E4 modified adenovirus, describedherein. The vaccine is administered to a patient, e.g., to treat cancer.In certain instances the patient is selected, e.g., using a companiondiagnostic or a commonly use cancer gene panel NGS assay such asFoundationOne, FoundationOne CDx, Guardant 360, Guardant OMNI, or MSKIMPACT. Exemplary patient selection criteria are described below.

Patient Selection

Patient selection for shared neoantigen vaccination is performed byconsideration of tumor gene expression, somatic mutation status, andpatient HLA type. Specifically, a patient is considered eligible for thevaccine therapy if:

-   -   (a) the patient carries an HLA allele predicted or known to        present an epitope included in a vaccine and the patient tumor        expresses a gene with the epitope sequence, or    -   (b) the patient carries an HLA allele predicted or known to        present an epitope included in a vaccine, and the patient tumor        carries the mutation giving rise to the epitope sequence, or    -   (c) Same as (b), but also requiring that the patient tumor        expresses the gene with the mutation above a certain threshold        (e.g., 1 TPM or 10 TPM), or    -   (d) Same as (b), but also requiring that the patient tumor        expresses the mutation above a certain threshold (e.g., at least        1 mutated read observed at the level of RNA)    -   (e) Same as (b), but also requiring both additional criteria        in (c) and (d)    -   (f) Any of the above, but also optionally requiring that loss of        the presenting HLA allele is not detected in the tumor

Gene expression is measured at the RNA or protein level by any of theestablished methods including RNASeq, microarray, PCR, Nanostring, ISH,Mass spectrometry, or IHC. Thresholds for positivity of gene expressionis established by several methods, including: (1) predicted probabilityof presentation of the epitope by the HLA allele at various geneexpression levels, (2) correlation of gene expression and HLA epitopepresentation as measured by mass spectrometry, and/or (3) clinicalbenefits of vaccination attained for patients expressing the genes atvarious levels. Patient selection is further extended to requirepositivity for greater than 1 epitope, for examples, at least 2, 3, 4 or5 epitopes included in the vaccine.

Somatic mutational status is assessed by any of the established methods,including exome sequencing (NGS DNASeq), targeted exome sequencing(panel of genes), transcriptome sequencing (RNASeq), Sanger sequencing,PCR-based genotyping assays (e.g., Taqman or droplet digital PCR),Mass-spectrometry based methods (e.g., by Sequenom), or any other methodknown to those skilled in the art.

Additional new shared neoantigens are identified using any of themethods described, e.g., by mass spectrometry. These newly identifiedshared neoantigens are incorporated into the vaccine cassettes describedherein.

Previously validated neoantigens are additionally validated as beingpresented by additional HLA alleles and informs neoantigen selection forthe vaccine cassette and/or expands the potential treatable population.

Inclusions of a new neoantigen enables the broadening of addressabletumor type (e.g., EGFR mutated NSCLC) or inclusion of patients with anew tumor type.

Certain Sequences

Vectors, cassettes, and antibodies referred to herein are describedbelow and referred to by SEQ ID NO.

Full-Length ChAdVC68 sequence “ChAdV68.5WTnt”(SEQ ID NO: 1); AC_000011.1  sequence with corresponding ATCC VR-594 nucleotides substituted at five positions; W at position 6 = A or T CCATCWTCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGATGAGGCACCTGAGAGACCTGCCCGATGAGAAAATCATCATCGCTTCCGGGAACGAGATTCTGGAACTGGTGGTAAATGCCATGATGGGCGACGACCCTCCGGAGCCCCCCACCCCATTTGAGACACCTTCGCTGCACGATTTGTATGATCTGGAGGTGGATGTGCCCGAGGACGATCCCAATGAGGAGGCGGTAAATGATTTTTTTAGCGATGCCGCGCTGCTAGCTGCCGAGGAGGCTTCGAGCTCTAGCTCAGACAGCGACTCTTCACTGCATACCCCTAGACCCGGCAGAGGTGAGAAAAAGATCCCCGAGCTTAAAGGGGAAGAGATGGACTTGCGCTGCTATGAGGAATGCTTGCCCCCGAGCGATGATGAGGACGAGCAGGCGATCCAGAACGCAGCGAGCCAGGGAGTGCAAGCCGCCAGCGAGAGCTTTGCGCTGGACTGCCCGCCTCTGCCCGGACACGGCTGTAAGTCTTGTGAATTTCATCGCATGAATACTGGAGATAAAGCTGTGTTGTGTGCACTTTGCTATATGAGAGCTTACAACCATTGTGTTTACAGTAAGTGTGATTAAGTTGAACTTTAGAGGGAGGCAGAGAGCAGGGTGACTGGGCGATGACTGGTTTATTTATGTATATATGTTCTTTATATAGGTCCCGTCTCTGACGCAGATGATGAGACCCCCACTACAAAGTCCACTTCGTCACCCCCAGAAATTGGCACATCTCCACCTGAGAATATTGTTAGACCAGTTCCTGTTAGAGCCACTGGGAGGAGAGCAGCTGTGGAATGTTTGGATGACTTGCTACAGGGTGGGGTTGAACCTTTGGACTTGTGTACCCGGAAACGCCCCAGGCACTAAGTGCCACACATGTGTGTTTACTTGAGGTGATGTCAGTATTTATAGGGTGTGGAGTGCAATAAAAAATGTGTTGACTTTAAGTGCGTGGTTTATGACTCAGGGGTGGGGACTGTGAGTATATAAGCAGGTGCAGACCTGTGTGGTTAGCTCAGAGCGGCATGGAGATTTGGACGGTCTTGGAAGACTTTCACAAGACTAGACAGCTGCTAGAGAACGCCTCGAACGGAGTCTCTTACCTGTGGAGATTCTGCTTCGGTGGCGACCTAGCTAGGCTAGTCTACAGGGCCAAACAGGATTATAGTGAACAATTTGAGGTTATTTTGAGAGAGTGTTCTGGTCTTTTTTGACGCTCTTAACTTGGGCCATCAGTCTCACTTTAACCAGAGGATTTCGAGAGCCCTGATTTTACTACTCCTGGCAGAACCACTGCAGCAGTAGCCTTTTTTGCTTTTATTCTTGACAAATGGAGTCAAGAAACCCATTTCAGCAGGGATTACCAGCTGGATTTCTTAGCAGTAGCCTGAGGATCCTGAATCTCCAGGAGAGTCCCAGGGCACGCCAACGTCGCCAGCAGCAGCAGCAGGAGGAGGATCAAGAAGAGAACCCGAGAGCCGGCCTGGACCCTCCGGCGGAGGAGGAGGAGTAGCTGACCTGTTTCCTGAACTGCGCCGGGTGCTGACTAGGTCTTCGAGTGGTCGGGAGAGGGGGATTAAGCGGGAGAGGCATGATGAGACTAATCACAGAACTGAACTGACTGTGGGTCTGATGAGTCGCAAGCGCCCAGAAACAGTGTGGTGGCATGAGGTGCAGTCGACTGGCACAGATGAGGTGTCGGTGATGCATGAGAGGTTTTCTCTAGAACAAGTCAAGACTTGTTGGTTAGAGCCTGAGGATGATTGGGAGGTAGCCATCAGGAATTATGCCAAGCTGGCTCTGAGGCCAGACAAGAAGTACAAGATTACTAAGCTGATAAATATCAGAAATGCCTGCTACATCTCAGGGAATGGGGCTGAAGTGGAGATCT GTCTCCAGGAAAGGGTGGCTTTCAGATGCTGCATGATGAATATGTACCCGGGAGTGGTGGGCATGGATGGGGTTACCTTTATGAACATGAGGTTCAGGGGAGATGGGTATAATGGCACGGTCTTTATGGCCAATACCAAGCTGACAGTCCATGGCTGCTCCTTCTTTGGGTTTAATAACACCTGCATCGAGGCCTGGGGTCAGGTCGGTGTGAGGGGCTGCAGTTTTTTCAGCCAACTGGATGGGGGTCGTGGGCAGGACCAAGAGTATGCTGTCCGTGAAGAAATGCTTGTTTGAGAGGTGCCACCTGGGGGTGATGAGCGAGGGCGAAGCCAGAATCCGCCACATGCGCCTCTACCGAGACGGGCTGCTTTGTGCTGTGCAAGGGCAATGCTAAGATCAAGCATAATATGATCTGTGGAGCCTCGGACGAGCGCGGCTACCAGATGCTGACCTGCGCCGGCGGGAACAGCCATATGCTGGCCACCGTACATGTGGCTTCCCATGCTCGCAAGCCCTGGCCCGAGTTCGAGCACAATGTCATGACCAGGTGCAATATGCATCTGGGGTCCCGCCGAGGCA TGTTCATGCCCTACCAGTGCAACCTGAATTATGTGAAGGTGCTGCTGGAGCCCGATGCCATGTCCAGAGTGAG CCTGACGGGGGTGTTTGACATGAATGTGGAGGTGTGGAAGATTCTGAGATATGATGAATCCAAGACCAGGTG CCGAGCCTGCGAGTGCGGAGGGAAGCATGCCAGTTCCAGCCCGTGTGTGTGGATGTGACGGAGGACCTGCG ACCCGATCATTTGGTGTTGCCCTGCACCGGGACGGAGTTCGGTTCCAGCGGGGAAGAATCTGACTAGAGTGA GTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGAACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTACCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCgGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTICCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCCCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGCGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATTCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTTGGCCTGGAGGCTAAGCGAACGGGTTGGCCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTITTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGAT GGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCACACCATCAACTTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGECCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCcGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCtGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCaATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGA.CCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGICGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTICGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCACCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATITCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTTCTGCAAGCTTCCTGCAGAAGAACTCAAGGGTCTGTGGACCGGGITCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAACUTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAATGTCCCATGGTGGCGCAGCTGACCTAGCTCGGCTTCGACACCTGGACCACTGCCGCCGCTTCCGCTGCTTCGCTCGGGATCTCGCCGAGTTTGCCTACTTTGAGCTGCCCGAGGAGCACCCTCAGGGCCCGGCCCACGGAGTGCGATCGTCGTCGAAGGGGGCCTCGACTCCCACCTGCTTCGGATCTTCAGCCAGCGTCCGATCCTGGTCGAGCGCGAGCAAGGACAGACCCTTCTGACTCTGTACTGCATCTGCAACCACCCCGGCCTGCATGAAAGTCTTTGTTGTCTGCTGTGTACTGAGTATAATAAAAGCTGAGATCAGCGACTACTCCGGACTTCCGTGTGTTCCTGAATCCATCAACCAGTCTTTGTTCTTCACCGGGAACGAGACCGAGCTCCAGCTCCAGTGTAAGCCCCACAAGAAGTACCTCACCTGGCTGTTCCAGGGCTCCCCGATCGCCGTTGTCAACCACTGCGACAACGACGGAGTCCTGCTGAGCGGCCCTGCCAACCTTACTITTTCCACCCGCAGAAGCAAGCTCCAGCTCTTCCAACCCTTCCTCCCCGGGACCTATCAGTGCGTCTCGGGACCCTGCCATCACACCTTCCACCTGATCCCGAATACCACAGCGTCGCTCCCCGCTACTAACAACCAAACTAACCTCCACCAACGCCACCGTCGCGACCTTTCTGAATCTAATACTACCACCCACACCGGAGGTGAGCTTCCGAGGTCAACCAACCTCTGGGATTTACTACGGCCCCTGGGAGGTGGTTGGGTTAATAGCGCTAGGCCTAGTTGCGGGTGGGCTTTTGGTTCTCTGCTACCTATACCTCCCTTGCTGTTCGTACTTAGTGGTGCTGTGTTGCTGGTTTAAGAAATGGGGAAGATCACCCTAGTGAGCTGCGGTGCGCTGGFGGCGGTGTTGCTTTCGATTGTGGGACTGGGCGGTGCGGCTGTAGTGAAGGAGAAGGCCGATCCCTGCTTGCATTTCAATCCCAACAAATGCCAGCTGAGTTTTCAGCCCGATGGCAATCGGTGCGCGGTACTGATCAAGTGCGGATGGGAATGCGAGAACGTGAGAATCGAGTACAATAACAAGACTCGGAACAATACTCTCGCGTCCGTGTGGCAGCCCGGGGACCCCGAGTGGTACACCGTCTCTGTCCCCGGTGCTGACGGCTCCCCGCGCACCGTGAATAATACTTTCATTTTTGCGCACATGTGCGACACGGTCATGTGGATGAGCAAGCAGTACGATATGTGGCCCCCCACGAAGGAGAACATCGTGGTCTTCTCCATCGCTTACAGCCTGTGCACGGCGCTAATCACCGCTATCGTGTGCCTGAGCATTCACATGCTCATCGCTATTCGCCCCAGAAATAATGCCGAAAAAGAAAAACAGCCATAACGTTTTTTTTCACACCTTTTTCAGACCATGGCCTCTGTTAAATTTTTGCTTTTATTTGCCAGTCTCATTGCCGTCATTCATGGAATGAGTAATGAGAAAATTACTATTTACACTGGCACTAATCACACATTGAAAGGTCCAGAAAAAGCCACAGAAGTTTCATGGTATTGTTATTTTAATGAATCAGATGTATCTACTGAACTCTGTGGAAACAATAACAAAAAAAATGAGAGCATTACTCTCATCAAGTTTCAATGTGGATCTGACTTAACCCTAATTAACATCACTAGAGACTATGTAGGTATGTATTATGGAACTACAGCAGGCATTTCGGACATGGAATTTTATCAAGTTTCTGTGTCTGAACCCACCACGCCTAGAATGACCACAACCACAAAAACTACACCTGTTACCACTATGCAGCTCACTACCAATAACATTTTTGCCATGCGTCAAATGGTCAACAATAGCACTCAACCCACCCCACCCAGTGAGGAAATTCCCAAATCCATGATTGGCATTATTGTTGCTGTAGTTGGTGTGCATGTTGATCATCGCCTTGTGCATGGTGTACTATGCCTTCTGCTACAGAAAGCACAGACTGAACGACAAGCTGGAACACTTACTAAGTGTTGAATTTTAATTTTTTAGAACCATGAAGATCCTAGGCCTTTTAATTTTTTCTATCATTACCTCTGCTCTATGCAATTCTGACAATGAGGACGTTACTGTCGTTGTCGGATCAAATTATACACTGAAAGGTCCAGCGAAGGGTATGCTTTCGTGGTATTGCTATTTTGGATCTGACACTACAGAAACTGAATTATGCAATCTTAAGAATGGCAAAATTCAAAATTCTAAAATTAACAATTATATATGCAATGGTACTGATCTGATACTCCTCAATATCACGAAATCATATGCTGGCAGTTACACCTGCCCTGGAGATGATGCTGACAGTATGATTTTTTACAAAGTAACTGTTGTTGATCCCACTACTCCACCTCCACCCACCACAACTACTCACACCACACACACAGATCAAACCGCAGCAGAGGAGGCAGCAAAGTTAGCCTTGCAGGTCCAAGACAGTTCATTTGTTGGCATTACCCCTACACCTGATCAGCGGTGTCCGGGGCTGCTAGTCAGCGGCATTGTCGGTGTGCTTTCGGGATTAGCAGTCATAATCATCTGCATGTTCATTTTTGCTTGCTGCTATAGAAGGCTTTACCGACAAAAATCAGACCCACTGCTGAACCTCTATGTTTAATTTTTTCCAGAGTCATGAAGGCAGTTAGCGCTCTAGTTTTTTGTTCTTTGATTGGCATTGTTTTTTGCAATCCTATTCCTAAAGTTAGCTTTATTAAAGATGTGAATGTTACTGAGGGGGGCAATGTGACACTGGTAGGTGTAGAGGGTGCTGAAAACACCACCTGGACAAAATACCACCTCAATGGGTGGAAAGATATTTGCAATTGGAGTGTATTAGTTTATACATGTGAGGGAGTTAATCTTACCATTGTCAATGCCACCTCAGCTCAAAATGGTAGAATTCAAGGACAAAGTGTCAGTGTATCTAATGGGTATTTTACCCAACATACTTTTATCTATGACGTTAAAGTCATACCACTGCCTACGCCTAGCCCACCTAGCACTACCACACAGACAACCCACACTACACAGACAACCACATACAGTACATTAAATCAGCCTACCACCACTACAGCAGCAGAGGTTGCCAGCTCGTCTGGGGTCCGAGTGGCATTTTTGATGTGGGCCCCATCTAGCAGTCCCACTGCTAGTACCAATGAGCAGACTACTGAATTTTTGTCCACTGTCGAGAGCCACACCACAGCTACCTCCAGTGCCTTCTCTAGCACCGCCAATCTCTCCTCGCTTTCCTCTACACCAATCAGTCCCGCTACTACTCCTAGCCCCGCTCCTCTTCCCACTCCCCTGAAGCAAACAGACGGCGGCATGCAATGGCAGATCACCCTGCTCATTGTGATCGGGTTGGTCATCCTGGCCGTGTTGCTCTACTACATCTTCTGCCGCCGCATTCCCAACGCGCACCGCAAGCCGGTCTACAAGCCCATCATTGTCGGGCAGCCGGAGCCGCTTCAGGTGGAAGGGGGTCTAAGGAATCTTCTCTTCTCTTTTACAGTATGGTGATTGAACTATGATTCCTAGACAATTCTTGATCACTATTCTTATCTGCCTCCTCCAAGTCTGTGCCACCCTCGCTCTGGTGGCCAACGCCAGTCCAGACTGTATTGGGCCCTTCGCCTCCTACGTGCTCTTTGCCTTCACCACCTGCATCTGCTGCTGTAGCATAGTCTGCCTGCTTATCACCTTCTTCCAGTTCATTGACTGGATCTTTGIGCGCATCGCCTACCTGCGCCACCACCCCCAGTACCGCGACCAGCGAGTGGCGCGGCTGCTCAGGCTCCTCTGATAAGCATGCGGGCTCTGCTACTTCTCGCGCTTCTGCTGTTAGTGCTCCCCCGTCCCGTCGACCCCCGGTCCCCCACCCAGTCCCCCGAGGAGGTCCGCAAATGCAAATTCCAAGAACCCTGGAAATTCCTCAAATGCTACCGCCAAAAATCAGACATGCATCCCAGCTGGATCATGATCATTGGGATCGTGAACATTCTGGCCTGCACCCTCATCTCCTTTGTGATTTACCCCTGCTTTGACTTTGGTTGGAACTCGCCAGAGGCGCTCTATCTCCCGCCTGAACCTGACACACCACCACAGCAACCTCAGGCACACGCACTACCACCACTACAGCCTAGGCCACAATACATGCCCATATTAGACTATGAGGCCGAGCCACAGCGACCCATGCTCCCCGCTATTAGTTACTTCAATCTAACCGGCGGAGATGACTGACCCACTGGCCAACAACAACGTCAACGACCTTCTCCTGGACATGGACGGCCGCGCCTCGGAGCAGCGACTCGCCCAACTTCGCATTCGCCAGCAGCAGGAGAGAGCCGTCAAGGAGCTGCAGGATGCGGTGGCCATCCACCAGTGCAAGAGAGGCATCTTCTGCCTGGTGAAACAGGCCAAGATCTCCTACGAGGTCACTCCAAACGACCATCGCCTCTCCTACGAGCTCCTGCAGCAGCGCCAGAAGTTCACCTGCCTGGTCGGAGTCAACCCCATCGTCATCACCCAGCAGTCTGGCGATACCAAGGGGTGCATCCACTGCTCCTGCGACTCCCCCGACTGCGTCCACACTCTGATCAAGACCCTCTGCGGCCTCCGCGACCTCCTCCCCATGAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTTCAAGCTGCTTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCACATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGGGGGAGGGAAGAACAGGAAGAACCATGATTAACTTTTAATCCAAACGGTCTCGGAGTACTTCAAAATGAAGATCGCGGAGATGGCACCTCTCGCCCCCGCTGTGTTGGTGGAAAATAACAGCCAGGTCAAAGGTGATACGGTTCTCGAGATGTTCCACGGTGGCTTCCAGCAAAGCCTCCACGCGCACATCCAGAAACAAGACAATAGCGAAAGCGGGAGGGTICTCTAATTCCTCAATCATCATGTTACACTCCTGCACCATCCCCAGATAATTTTCATTTTTCCAGCCTTGAATGATTCGAACTAGTTCcTGAGGTAAATCCAAGCCAGCCATGATAAAGAGCTCGCGCAGAGCGCCCTCCACCGGCATTCTTAAGCACACCCTCATAATTCCAAGATATTCTGCTCCTGGTTCACCTGCAGCAGATTGACAAGCGGAATATCAAAATCTCTGCCGCGATCCCTGAGCTCCTCCCTCAGCAATAACTGTAAGTACTCTTTCATATCCTCTCCGAAATTTTTAGCCATAGGACCACCAGGAATAAGATTAGGGCAAGCCACAGTACAGATAAACCGAAGTCCTCCCCAGTGAGCATTGCCAAATGCAAGACTGCTATAAGCATGCTGGCTAGACCCGGTGATATCTTCCAGATAACTGGACAGAAAATCGCCCAGGCAATTTTTAAGAAAATCAACAAAAGAAAAATCCTCCAGGTGGACGTTTAGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGCUCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATGG Tremelimumab VL (SEQ ID NO: 16)Tremelimumab VH (SEQ ID NO: 17) Tremelimumab VH CDR (SEQ ID NO: 18)Tremelimumab VH CDR2 (SEQ ID NO: 19)Tremelimumab VH CDR3 (SEQ ID NO: 20)Tremelimumab VL CDR1 (SEQ ID NO: 21)Tremelimumab VL CDR2 (SEQ ID NO: 22)Tremelimumab VL CDR3 (SEQ ID NO: 23)Durvalumab (MEDI4736) VL (SEQ ID NO: 24) MEDI4736 VH (SEQ ID NO: 25)MEDI4736 VH CDR1 (SEQ ID NO: 26) MEDI4736 VH CDR2 (SEQ ID NO: 27)MEDI4736 VH CDR3 (SEQ ID NO: 28) MEDI4736 VL CDR1 (SEQ ID NO: 29)MEDI4736 VL CDR2 (SEQ ID NO: 30) MEDI4736 VL CDR3 (SEQ ID NO: 31)UbA76-25merPDTT nucleotide (SEQ ID NO: 32)UbA76-25merPDTT polypeptide (SEQ ID NO: 33)MAG-25merPDTT nucleotide (SEQ ID NO: 34)MAG-25merPDTT polypeptide (SEQ ID NO: 35)Ub7625merPDTT_NoSFL nucleotide (SEQ ID NO: 36)Ub7625merPDTT_NoSFL polypeptide (SEQ ID NO: 37)ChAdV68.5WTnt.MAG25mer (SEQ ID NO: 2); AC_000011.1 with E1 (nt 577 to 3403)and E3 (nt 27, 125-31, 825) sequences deleted; corresponding ATCC VR-594nucleotides substituted at five positions; model neoantigen cassetteunder the control of the CMV promoter/enhancer inserted in place of deleted E1;SV40 polyA 3′ of cassetteVenezuelan equine encephalitis virus [VEE] (SEQ ID NO: 3) GenBank: L01442.2VEE-MAG25mer (SEQ ID NO: 4); contains MAG-25merPDTT nucleotide (bases 30-1755)Venezuelan equine encephalitis virus strain TC-83 [TC-83] (SEQ ID NO: 5)GenBank: L01443.1VEE Delivery Vector (SEQ ID NO: 6); VEE genome with nucleotides 7544-11175deleted [alphavirus structural proteins removed]TC-83 Delivery Vector(SEQ ID NO: 7); TC-83 genome with nucleotides 7544-11175deleted [alphavirus structural proteins removed]VEE Production Vector (SEQ ID NO: 8); VEE genome with nucleotides 7544-11175deleted, plus 5′ T7-promoter, plus 3′ restriction sitesTC-83 Production Vector(SEQ ID NO: 9); TC-83 genome with nucleotides 7544-11175 deleted, plus 5′ T7-promoter, plus 3′ restriction sitesVEE-UbAAY (SEQ ID NO: 14); VEE delivery vector with MHC class I mouse tumorepitopes SIINFEKL and AH1-A5 insertedVEE-Luciferase (SEQ ID NO: 15); VEE delivery vector with luciferase geneinserted at 7545 ubiquitin (SEQ ID NO: 38) > UbG76 0-228Ubiquitin A76 (SEQ ID NO: 39) > UbA76 0-228HLA-A2 (MHC class I) signal peptide (SEQ ID NO 40) > MHC SignalPep 0-78HLA-A2 (MHC class I) Trans Membrane domain (SEQ ID NO: 41) > HLA A2 TMDomain 0-201 IgK Leader Seq (SEQ ID NO: 42) > IgK Leader Seq 0-60Human DC-Lamp (SEQ ID NO: 43) > HumanDCLAMP 0-3178Mouse LAMP1 (SEQ ID NO: 44) > MouseLamp1 0-1858Human Lamp1 cDNA (SEQ ID NO: 45) > Human Lamp1 0-2339Tetanus toxoid nulceic acid sequence (SEQ ID NO: 46)Tetanus toxoid amino acid sequence (SEQ ID NO: 47)PADRE nulceotide sequence (SEQ ID NO: 48)PADRE amino acid sequence (SEQ ID NO: 49)WPRE (SEQ ID NO: 50) > WPRE 0-593IRES (SEQ ID N0: 51) > eGFP_IRES_SEAP_Insert 1746-2335GFP (SEQ ID NO: 52) SEAP (SEQ ID NO: 53)Firefly Luciferase (SEQ ID NO: 54) FMDV 2A (SEQ ID NO: 55)ChAdV68-MAG-E4deleted (SEQ ID NO: 57)CATCaTCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATgacattgattattgactagttGttaaTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGgTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAgcTCGTTTAGTGAACCGTCAGATCGCCTGGAACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGccaccATGGCCGGGATGTTCCAGGCACTGTCCGAAGGCTGCACACCCTATGATATTAACCAGATGCTGAATGTCCTGGGAGACCACCAGGTCTCTGGCCTGGAGCAGCTGGAGAGCATCATCAACTTCGAGAAGCTGACCGAGTGGACAAGCTCCAATGTGATGCCTATCCTGTCCCCACTGACCAAGGGCATCCTGGGCTTCGTGTTTACCCTGACAGTGCCTTCTGAGCGGGGCCTGTCTTGCATCAGCGAGGCAGACGCAACCACACCAGAGTCCGCCAATCTGGGCGAGGAGATCCTGTCTCAGCTGTACCTGTGGCCCCGGGTGACATATCACTCCCCTTCTTACGCCTATCACCAGTTCGAGCGGAGAGCCAAGTACAAGAGACACTTCCCAGGCTTTGGCCAGTCTCTGCTGTTCGGCTACCCCGTGTACGTGTTCGGCGATTGCGTGCAGGGCGACTGGGATGCCATCCGGTTTAGATACTGCGCACCACCTGGATATGCACTGCTGAGGTGTAACGACACCAATTATTCCGCCCTGCTGGCAGTGGGCGCCCTGGAGGGCCCTCGCAATCAGGATTGGCTGGGCGTGCCAAGGCAGCTGGTGACACGCATGCAGGCCATCCAGAACGCAGGCCTGTGCACCCTGGTGGCAATGCTGGAGGAGACAATCTTCTGGCTGCAGGCCTTTCTGATGGCCCTGACCGACAGCGGCCCCAAGACAAACATCATCGTGGATTCCCAGTACGTGATGGGCATCTCCAAGCCTTCTTTCCAGGAGTTTGTGGACTGGGAGAACGTGAGCCCAGAGCTGAATTCCACCGATCAGCCATTCTGGCAGGCAGGAATCCTGGCAAGGAACCTGGTGCCTATGGTGGCCACAGTGCAGGGCCAGAATCTGAAGTACCAGGGCCAGAGCCTGGTCATCAGCGCCTCCATCATCGTGTTTAACCTGCTGGAGCTGGAGGGCGACTATCGGGACGATGGCAACGTGTGGGTGCACACCCCACTGAGCCCCAGAACACTGAACGCCTGGGTGAAGGCCGTGGAGGAGAAGAAGGGCATCCCAGTGCACCTGGAGCTGGCCTCCATGACCAATATGGAGCTGATGTCTAGCATCGTGCACCAGCAGGTGAGGACATACGGACCCGTGTTCATGTGCCTGGGAGGCCTGCTGACCATGGTGGCAGGAGCCGTGTGGCTGACAGTGCGGGTGCTGGAGCTGTTCAGAGCCGCCCAGCTGGCCAACGATGTGGTGCTGCAGATCATGGAGCTGTGCGGAGCAGCCTTTCGCCAGGTGTGCCACACCACAGTGCCATGGCCCAATGCCTCCCTGACCCCCAAGTGGAACAATGAGACAACACAGCCTCAGATCGCCAACTGTAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTATAGCGTGAGGGATACCCTGTGGCCCCGCGTGACATACCACATGAATAAGTACGCCTATCACATGCTGGAGAGGCGCGCCAAGTATAAGAGAGGCCCTGGCCCAGGCGCAAAGTTTGTGGCAGCATGGACCCTGAAGGCCGCCGCCGGCCCCGGCCCCGGCCAGTATATCAAGGCTAACAGTAAGTTCATTGGAATCACAGAGCTGGGACCCGGACCTGGATAATGAGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTtCCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCgGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCcGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCtGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCaATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATGChAdV68-GFP-E4deleted (SEQ ID NO: 58); Bold italicized = GFP transgeneCATCaTCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGATAACAGGGTAATgacattgattattgactagttGttaaTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGgTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAgcTCGTTTAGTGAACCGTCAGATCGCCTGGAACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAGCGATCGCGccacc

tgaGTTTAAACTCCCATTTAAATGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATAACTATAACGGTCCTAAGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTtCCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCgGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCcGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCtGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCaATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATGChAdV68-Empty-E4deleted (SEQ ID NO: 59)CATCTTCAATAATATACCTCAAACTTTTTGTGCGCGTTAATATGCAAATGAGGCGTTTGAATTTGGGGAGGAAGGGCGGTGATTGGTCGAGGGATGAGCGACCGTTAGGGGCGGGGCGAGTGACGTTTTGATGACGTGGTTGCGAGGAGGAGCCAGTTTGCAAGTTCTCGTGGGAAAAGTGACGTCAAACGAGGTGTGGTTTGAACACGGAAATACTCAATTTTCCCGCGCTCTCTGACAGGAAATGAGGTGTTTCTGGGCGGATGCAAGTGAAAACGGGCCATTTTCGCGCGAAAACTGAATGAGGAAGTGAAAATCTGAGTAATTTCGCGTTTATGGCAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGACCGATTACGTGGGGGTTTCGATTACCGTGTTTTTCACCTAAATTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAGGTGTCAGCTGATCGCCAGGGTATTTAAACCTGCGCTCTCCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTTCTCCTCCGCGCCGCGAGTCAGATCTACACTTTGAAAGTAGGGataaGGTAGCGAGTGAGTAGTGTTCTGGGGCGGGGGAGGACCTGCATGAGGGCCAGAATAACTGAAATCTGTGCTTTTCTGTGTGTTGCAGCAGCATGAGCGGAAGCGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTCTCCCCTCCTGGGCGGGAGTGCGTCAGAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTGCAGCCCGCGAACTCTTCAACCCTGACCTATGCAACCCTGAGCTCTTCGTCGTTGGACGCAGCTGCCGCCGCAGCTGCTGCATCTGCCGCCAGCGCCGTGCGCGGAATGGCCATGGGCGCCGGCTACTACGGCACTCTGGTGGCCAACTCGAGTTCCACCAATAATCCCGCCAGCCTGAACGAGGAGAAGCTGTTGCTGCTGATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCTCAGCTGCAGGAGCAGACGCGGGCCGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGGTTGTTGATTTTAACACAGAGTCTGAATCTTTATTTGATTTTTCGCGCGCGGTAGGCCCTGGACCACCGGTCTCGATCATTGAGCACCCGGTGGATCTTTTCCAGGACCCGGTAGAGGTGGGCTTGGATGTTGAGGTACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCTCCATTGCAGGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGGCGCAGGGCATGGTGTTGCACAATATCTTTGAGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAATCTGTTGAGCTGGGAGGGATGCATGCGGGGGGAGATGAGGTGCATCTTGGCCTGGATCTTGAGATTGGCGATGTTACCGCCCAGATCCCGCCTGGGGTTCATGTTGTGCAGGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTATCATGCAACTTGGAAGGGAAGGCGTGAAAGAATTTGGCGACGCCTTTGTGCCCGCCCAGGTTTTCCATGCACTCATCCATGATGATGGCGATGGGCCCGTGGGCGGCGGCCTGGGCAAAGACGTTTCGGGGGTCGGACACATCATAGTTGTGGTCCTGGGTGAGGTCATCATAGGCCATTTTAATGAATTTGGGGCGGAGGGTGCCGGACTGGGGGACAAAGGTACCCTCGATCCCGGGGGCGTAGTTCCCCTCACAGATCTGCATCTCCCAGGCTTTGAGCTCGGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAACACGGTTTCCGGGGCGGGGGAGATGAGCTGGGCCGAAAGCAAGTTCCGGAGCAGCTGGGACTTGCCGCAGCCGGTGGGGCCGTAGATGACCCCGATGACCGGCTGCAGGTGGTAGTTGAGGGAGAGACAGCTGCCGTCCTCCCGGAGGAGGGGGGCCACCTCGTTCATCATCTCGCGCACGTGCATGTTCTCGCGCACCAGTTCCGCCAGGAGGCGCTCTCCCCCCAGGGATAGGAGCTCCTGGAGCGAGGCGAAGTTTTTCAGCGGCTTGAGTCCGTCGGCCATGGGCATTTTGGAGAGGGTTTGTTGCAAGAGTTCCAGGCGGTCCCAGAGCTCGGTGATGTGCTCTACGGCATCTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGACGGCTGCGGGAGTAGGGCACCAGACGATGGGCGTCCAGCGCAGCCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTGAAGGGGTGCGCGCCGGGCTGGGCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAAAACCGCTCCCGATCGGCGCCCTGCGCGTCGGCCAGGTAGCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTTACCTTTGGAAGTCTGCCCGCAGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGGGGGCGTAGGCGTCCGCGCCGCAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGGTCGGGGTCAAAAACCAGTTTCCCGCCGTTCTTTTTGATGCGTTTCTTACCTTTGGTCTCCATGAGCTCGTGTCCCCGCTGGGTGACAAAGAGGCTGTCCGTGTCCCCGTAGACCGACTTTATGGGCCGGTCCTCGAGCGGTGTGCCGCGGTCCTCCTCGTAGAGGAACCCCGCCCACTCCGAGACGAAAGCCCGGGTCCAGGCCAGCACGAAGGAGGCCACGTGGGACGGGTAGCGGTCGTTGTCCACCAGCGGGTCCACCTTTTCCAGGGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTAGGCCACGTGACCGGGGGTCCCGGCCGGGGGGGTATAAAAGGGTGCGGGTCCCTGCTCGTCCTCACTGTCTTCCGGATCGCTGTCCAGGAGCGCCAGCTGTTGGGGTAGGTATTCCCTCTCGAAGGCGGGCATGACCTCGGCACTCAGGTTGTCAGTTTCTAGAAACGAGGAGGATTTGATATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGACGATCTTTTTGTTGTCGAGCTTGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCTGGTTTTTTTCCTTGTCGGCGCGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGGAAGACGGTGGTCAGCTCGTCGGGCACGATTCTGACCTGCCAGCCCCGATTATGCAGGGTGATGAGGTCCACACTGGTGGCCACCTCGCCGCGCAGGGGCTCATTAGTCCAGCAGAGGCGTCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCATGACCTCGTCGGGGGGGTCGGCATCGATGGTGAAGATGCCGGGCAGGAGGTCGGGGTCAAAGTAGCTGATGGAAGTGGCCAGATCGTCCAGGGCAGCTTGCCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTGAGGGGCGTGCCCCAGGGCATGGGATGGGTAAGCGCGGAGGCGTACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCCTCGAGGATGCCGATGTAGGTGGGGTAGCAGCGCCCCCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGCCCCGGGCCCAGGTTGGTGCGACTGGGCTTTTCGGCGCGGTAGACGATCTGGCGGAAAATGGCATGCGAGTTGGAGGAGATGGTGGGCCTTTGGAAGATGTTGAAGTGGGCGTGGGGCAGTCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACGAGCTCGGCGGTGACTAGGACGTCCAGAGCGCAGTAGTCGAGGGTCTCCTGGATGATGTCATACTTGAGCTGTCCCTTTTGTTTCCACAGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCGAGGGGGAACCCGTCCTGATCTGCACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCGTAGGCCTGGGCGGCCTTGCGCAGGGAGGTGTGCGTGAGGGCGAAAGTGTCCCTGACCATGACCTTGAGGAACTGGTGCTTGAAGTCGATATCGTCGCAGCCCCCCTGCTCCCAGAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTTGAAGAGGATCTTGCCCGCGCGGGGCATAAAGTTGCGAGTGATGCGGAAAGGTTGGGGCACCTCGGCCCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGATGTAGAGTTCCACGAATCGCGGACGGCCCTTGACGTGGGGCAGTTTCTTGAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGCGCCCAGTCGGCGAGATGGGGGTTGGCGCGGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGGTCCCGGTACTGACGGAACTGCTGCCCGACGGCCATTTTTTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCCCCGTGCCAGCGATCCCATTTGAGCTGGAGGGCGAGATCGAGGGCGAGCTCGACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAGCTGCTTGCCGAAGGACCCCATCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGCCGATGGGGAAGAACTGGATCTCCTGCCACCAATTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGCGCCGAACACTCGTGCTTGTGTTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTGTACCTGAGTTCCTTTGACGAGGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTCGTGCTGTACTACGTCGTGGTGGTCGGCCTGGCCCTCTTCTGCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGAGCGGGTCGGAGAGCGAGGACGAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGGCAGCGGCGGCGCGCGGTTGACTTGCAGGAGTTTTTCCAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGCCATTGGTGGCGACGTCGATGGCTTGCAGGGTCCCGTGCCCCTGGGGTGTGACCACCGTCCCCCGTTTCTTCTTGGGCGGCTGGGGCGACGGGGGCGGTGCCTCTTCCATGGTTAGAAGCGGCGGCGAGGACGCGCGCCGGGCGGCAGGGGCGGCTCGGGGCCCGGAGGCAGGGGCGGCAGGGGCACGTCGGCGCCGCGCGCGGGTAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGACGCGACGGTTGACGTCCTGGATCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGAGAGTTCGACAGAATCAATCTCGGTATCGTTGACGGCGGCCTGCCGCAGGATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCGATCTCGGTCATGAACTGCTCGATCTCCTCCTCTTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGAGATGCGGCCCATGAGCTGCGAGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAGACCACGACGCCCTCGGGATCGCgGGCGCGCATGACCACCTGGGCGAGGTTGAGCTCCACGTGGCGCGTGAAGACCGCGTAGTTGCAGAGGCGCTGGTAGAGGTAGTTGAGCGTGGTGGCGATGTGCTCGGTGACGAAGAAATACATGATCCAGCGGCGGAGCGGCATCTCGCTGACGTCGCCCAGCGCCTCCAAACGTTCCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGAGACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCCCCCGGGAGTTCCTCCACTTCCTCTTCTTCCTCCTCCACTAACATCTCTTCTACTTCCTCCTCAGGCGGCAGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCGGCGGCGCACGGGCAGACGGTCGATGAAGCGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCCCGTCCTCGCGGGGCCGCAGCGTGAAGACGCCGCCGCGCATCTCCAGGTGGCCGGGGGGGTCCCCGTTGGGCAGGGAGAGGGCGCTGACGATGCATCTTATCAATTGCCCCGTAGGGACTCCGCGCAAGGACCTGAGCGTCTCGAGATCCACGGGATCTGAAAACCGCTGAACGAAGGCTTCGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGGTCATGTTGGTTGGGAGCGGGGCGGGCGATGCTGCTGGTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCTTTGGGCCCGGCTTGCTGGATGCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTAGTAGTCCTGCATGAGCCGCTCCACGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATGCGCGTGAGCCCGAAGCCGCGCTGGGGCTGGACGAGCGCCAGGTCGGCGACGACGCGCTCGGCGAGGATGGCTTGCTGGATCTGGGTGAGGGTGGTCTGGAAGTCATCAAAGTCGACGAAGCGGTGGTAGGCTCCGGTGTTGATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGACGCACGAGCTCGTGGTACTTGAGGCGCGAGTAGGCGCGCGTGTCGAAGATGTAGTCGTTGCAGGTGCGCACCAGGTACTGGTAGCCGATGAGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCGGGCGCGAGGTCCTCGAGCATGGTGCGGTGGTAGCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGGAACTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCGTGAGGCGCGCGCAGTCGTGGATGCTCTATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAGGCTAAGCGAACGGGTTGGGCTGCGCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTATTGGCACTCCCGTCTCGACCCAAGCCTGCACCAACCCTCCAGGATACGGAGGCGGGTCGTTTTGCAACTTTTTTTTGGAGGCCGGATGAGACTAGTAAGCGCGGAAAGCGGCCGACCGCGATGGCTCGCTGCCGTAGTCTGGAGAAGAATCGCCAGGGTTGCGTTGCGGTGTGCCCCGGTTCGAGGCCGGCCGGATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAGACCCCATAGCCAGCCGACTTCTCCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTGTTTTTGCCAGATGCATCCCGTACTGCGGCAGATGCGCCCCCACCACCCTCCACCGCAACAACAGCCCCCTCCACAGCCGGCGCTTCTGCCCCCGCCCCAGCAGCAACTTCCAGCCACGACCGCCGCGGCCGCCGTGAGCGGGGCTGGACAGAGTTATGATCACCAGCTGGCCTTGGAAGAGGGCGAGGGGCTGGCGCGCCTGGGGGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGCCTACGTGCCCAAGCAGAACCTGTTCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCGGCCCGGTTCCACGCGGGGCGGGAGCTGCGGCGCGGCCTGGACCGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCCGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCAAAAATCCTTCAACAACCACGTGCGCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGCTGGAGGCCATCGTGCAGAACCCCACCAGCAAGCCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCATAGTCGGGACAACGAAGCGTTCAGGGAGGCGCTGCTGAATATCACCGAGCCCGAGGGCCGCTGGCTCCTGGACCTGGTGAACATTCTGCAGAGCATCGTGGTGCAGGAGCGCGGGCTGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTCTCGGTGCTGAGTTTGGGCAAGTACTACGCTAGGAAGATCTACAAGACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATGACCCTGAAAGTGCTGACCCTGAGCGACGATCTGGGGGTGTACCGCAACGACAGGATGCACCGTGCGGTGAGCGCCAGCAGGCGGCGCGAGCTGAGCGACCAGGAGCTGATGCATAGTCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCTACTTTGACATGGGCGCGGACCTGCACTGGCAGCCCAGCCGCCGGGCCTTGGAGGCGGCGGCAGGACCCTACGTAGAAGAGGTGGACGATGAGGTGGACGAGGAGGGCGAGTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAACAACAACAGCCACCTCCTGATCCCGCGATGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCAACGCATCATGGCGCTGACGACCCGCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGGAGGCCGTGGTGCCCTCGCGCTCCAACCCCACGCACGAGAAGGTCCTGGCCATCGTGAACGCGCTGGTGGAGAACAAGGCCATCCGCGGCGACGAGGCCGGCCTGGTGTACAACGCGCTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGACCAACCTGGACCGCATGGTGACCGACGTGCGCGAGGCCGTGGCCCAGCGCGAGCGGTTCCACCGCGAGTCCAACCTGGGATCCATGGTGGCGCTGAACGCCTTCCTCAGCACCCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATCAGCGCCCTGCGCCTGATGGTGACCGAGGTGCCCCAGAGCGAGGTGTACCAGTCCGGGCCGGACTACTTCTTCCAGACCAGTCGCCAGGGCTTGCAGACCGTGAACCTGAGCCAGGCTTTCAAGAACTTGCAGGGCCTGTGGGGCGTGCAGGCCCCGGTCGGGGACCGCGCGACGGTGTCGAGCCTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCCCCCTTCACGGACAGCGGCAGCATCAACCGCAACTCGTACCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATCGGCCAGGCGCACGTGGACGAGCAGACCTACCAGGAGATCACCCACGTGAGCCGCGCCCTGGGCCAGGACGACCCGGGCAACCTGGAAGCCACCCTGAACTTTTTGCTGACCAACCGGTCGCAGAAGATCCCGCCCCAGTACGCGCTCAGCACCGAGGAGGAGCGCATCCTGCGTTACGTGCAGCAGAGCGTGGGCCTGTTCCTGATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGAGCCCAGCATGTACGCCAGCAACCGCCCGTTCATCAATAAACTGATGGACTACTTGCATCGGGCGGCCGCCATGAACTCTGACTATTTCACCAACGCCATCCTGAATCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGACCCCAATGACGGGTTCCTGTGGGACGATGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTGGAAGAAGGAAGGCAGCGACCGACGCCCGTCCTCGGCGCTGTCCGGCCGCGAGGGTGCTGCCGCGGCGGTGCCCGAGGCCGCCAGTCCTTTCCCGAGCTTGCCCTTCTCGCTGAACAGTATCCGCAGCAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGCGAAGAGGAGTACTTGAATGACTCGCTGTTGAGACCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAAAGCCTGGTGGACAAGATGAGCCGCTGGAAGACGTATGCGCAGGAGCACAGGGACGATCCCCGGGCGTCGCAGGGGGCCACGAGCCGGGGCAGCGCCGCCCGTAAACGCCGGTGGCACGACAGGCAGCGGGGACAGATGTGGGACGATGAGGACTCCGCCGACGACAGCAGCGTGTTGGACTTGGGTGGGAGTGGTAACCCGTTCGCTCACCTGCGCCCCCGTATCGGGCGCATGATGTAAGAGAAACCGAAAATAAATGATACTCACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTTGTTGTATCTAGTATGATGAGGCGTGCGTACCCGGAGGGTCCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCGATGGCGGCGGCGGCGATGCAGCCCCCGCTGGAGGCTCCTTACGTGCCCCCGCGGTACCTGGCGCCTACGGAGGGGCGGAACAGCATTCGTTACTCGGAGCTGGCACCCTTGTACGATACCACCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGAACGACCACAGCAACTTCCTGACCACCGTGGTGCAGAACAATGACTTCACCCCCACGGAGGCCAGCACCCAGACCATCAACTTTGACGAGCGCTCGCGGTGGGGCGGCCAGCTGAAAACCATCATGCACACCAACATGCCCAACGTGAACGAGTTCATGTACAGCAACAAGTTCAAGGCGCGGGTGATGGTCTCCCGCAAGACCCCCAATGGGGTGACAGTGACAGAGGATTATGATGGTAGTCAGGATGAGCTGAAGTATGAATGGGTGGAATTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATCGACCTGATGAACAACGCCATCATCGACAATTACTTGGCGGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTTCGACACTAGGAACTTCAGGCTGGGCTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCTTTCCATCCCGATATTGTCTTGCTGCCCGGCTGCGGGGTGGACTTCACCGAGAGCCGCCTCAGCAACCTGCTGGGCATTCGCAAGAGGCAGCCCTTCCAGGAAGGCTTCCAGATCATGTACGAGGATCTGGAGGGGGGCAACATCCCCGCGCTCCTGGATGTCGACGCCTATGAGAAAAGCAAGGAGGATGCAGCAGCTGAAGCAACTGCAGCCGTAGCTACCGCCTCTACCGAGGTCAGGGGCGATAATTTTGCAAGCGCCGCAGCAGTGGCAGCGGCCGAGGCGGCTGAAACCGAAAGTAAGATAGTCATTCAGCCGGTGGAGAAGGATAGCAAGAACAGGAGCTACAACGTACTACCGGACAAGATAAACACCGCCTACCGCAGCTGGTACCTAGCCTACAACTATGGCGACCCCGAGAAGGGCGTGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACTGGTCGCTGCCCGACATGATGCAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCCGAGCTCCTGCCCGTCTACTCCAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCTTACGCACGTCTTCAACCGCTTCCCCGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTAAATGTCCATTCTCATCTCGCCCAGTAATAACACCGGTTGGGGCCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTCGCCAACGCTCCACGCAACACCCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGGTCGCGCACCACCGTCGACGACGTGATCGACCAGGTGGTGGCCGACGCGCGCAACTACACCCCCGCCGCCGCGCCCGTCTCCACCGTGGACGCCGTCATCGACAGCGTGGTGGCcGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCGCCCGGCGGCACCGGAGCACCCCCGCCATGCGCGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCCATGCTCAGGGCGGCCAGACGCGCGGCTTCAGGCGCCAGCGCCGGCAGGACCCGGAGACGCGCGGCCACGGCGGCGGCAGCGGCCATCGCCAGCATGTCCCGCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCGTGCGCACCCGCCCCCCTCGCACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGGATGTCCAAGCGCAAATTCAAGGAAGAGATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCTGCGGTGGTGAAGGAGGAAAGAAAGCCCCGCAAAATCAAGCGGGTCAAAAAGGACAAAAAGGAAGAAGAAAGTGATGTGGACGGATTGGTGGAGTTTGTGCGCGAGTTCGCCCCCCGGCGGCGCGTGCAGTGGCGCGGGCGGAAGGTGCAACCGGTGCTGAGACCCGGCACCACCGTGGTCTTCACGCCCGGCGAGCGCTCCGGCACCGCTTCCAAGCGCTCCTACGACGAGGTGTACGGGGATGATGATATTCTGGAGCAGGCGGCCGAGCGCCTGGGCGAGTTTGCTTACGGCAAGCGCAGCCGTTCCGCACCGAAGGAAGAGGCGGTGTCCATCCCGCTGGACCACGGCAACCCCACGCCGAGCCTCAAGCCCGTGACCTTGCAGCAGGTGCTGCCGACCGCGGCGCCGCGCCGGGGGTTCAAGCGCGAGGGCGAGGATCTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAGACCATGAAGGTGGACCCGGACGTGCAGCCCGAGGTCAAGGTGCGGCCCATCAAGCAGGTGGCCCCGGGCCTGGGCGTGCAGACCGTGGACATCAAGATTCCCACGGAGCCCATGGAAACGCAGACCGAGCCCATGATCAAGCCCAGCACCAGCACCATGGAGGTGCAGACGGATCCCTGGATGCCATCGGCTCCTAGTCGAAGACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAACTACGCGCTGCATCCTTCCATCATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCGCGGTCATACCAGCAGCCGCCGCCGCAAGACCACCACTCGCCGCCGCCGTCGCCGCACCGCCGCTGCAACCACCCCTGCCGCCCTGGTGCGGAGAGTGTACCGCCGCGGCCGCGCACCTCTGACCCTGCCGCGCGCGCGCTACCACCCGAGCATCGCCATTTAAACTTTCGCCtGCTTTGCAGATCAATGGCCCTCACATGCCGCCTTCGCGTTCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGGAACGGGATGCGTCGCCACCACCACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATCCCCATCATCGCCGCGGCGATCGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACACACTTGGAAACATCTTGTAATAAACCaATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTCGTAGACAGATGGAAGACATCAATTTTTCGTCCCTGGCTCCGCGACACGGCACGCGGCCGTTCATGGGCACCTGGAGCGACATCGGCACCAGCCAACTGAACGGGGGCGCCTTCAATTGGAGCAGTCTCTGGAGCGGGCTTAAGAATTTCGGGTCCACGCTTAAAACCTATGGCAGCAAGGCGTGGAACAGCACCACAGGGCAGGCGCTGAGGGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTCGATGGGCTCGCCTCGGGCATCAACGGGGTGGTGGACCTGGCCAACCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGTGCCGCCCGCCGGCTCCGTGGAGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGAGAAGCGACCCCGCCCCGATGCGGAGGAGACGCTGCTGACGCACACGGACGAGCCGCCCCCGTACGAGGAGGCGGTGAAACTGGGTCTGCCCACCACGCGGCCCATCGCGCCCCTGGCCACCGGGGTGCTGAAACCCGAAAAGCCCGCGACCCTGGACTTGCCTCCTCCCCAGCCTTCCCGCCCCTCTACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCCGGGGGCACCGCCCGCCCTCATGCGAACTGGCAGAGCACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGCTATTAAACCTACCGTAGCGCTTAACTTGCTTGTCTGTGTGTGTATGTATTATGTCGCCGCCGCCGCTGTCCACCAGAAGGAGGAGTGAAGAGGCGCGTCGCCGAGTTGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAGGACGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTTGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTCGTGCCCGTGGACCGCGAGGACAACACCTACTCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGCACCTACTTTGACATCCGCGGCGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACCGCCTACAACAGTCTGGCCCCCAAGGGAGCACCCAACACTTGTCAGTGGACATATAAAGCCGATGGTGAAACTGCCACAGAAAAAACCTATACATATGGAAATGCACCCGTGCAGGGCATTAACATCACAAAAGATGGTATTCAACTTGGAACTGACACCGATGATCAGCCAATCTACGCAGATAAAACCTATCAGCCTGAACCTCAAGTGGGTGATGCTGAATGGCATGACATCACTGGTACTGATGAAAAGTATGGAGGCAGAGCTCTTAAGCCTGATACCAAAATGAAGCCTTGTTATGGTTCTTTTGCCAAGCCTACTAATAAAGAAGGAGGTCAGGCAAATGTGAAAACAGGAACAGGCACTACTAAAGAATATGACATAGACATGGCTTTCTTTGACAACAGAAGTGCGGCTGCTGCTGGCCTAGCTCCAGAAATTGTTTTGTATACTGAAAATGTGGATTTGGAAACTCCAGATACCCATATTGTATACAAAGCAGGCACAGATGACAGCAGCTCTTCTATTAATTTGGGTCAGCAAGCCATGCCCAACAGACCTAACTACATTGGTTTCAGAGACAACTTTATCGGGCTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCTTCTCAGCTGAATGCTGTGGTTGACTTGCAAGACAGAAACACCGAGCTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCGGTATTTCAGTATGTGGAATCAGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAATCATGGTGTGGAGGATGAACTTCCCAACTATTGTTTCCCTCTGGATGCTGTTGGCAGAACAGATACTTATCAGGGAATTAAGGCTAATGGAACTGATCAAACCACATGGACCAAAGATGACAGTGTCAATGATGCTAATGAGATAGGCAAGGGTAATCCATTCGCCATGGAAATCAACATCCAAGCCAACCTGTGGAGGAACTTCCTCTACGCCAACGTGGCCCTGTACCTGCCCGACTCTTACAAGTACACGCCGGCCAATGTTACCCTGCCCACCAACACCAACACCTACGATTACATGAACGGCCGGGTGGTGGCGCCCTCGCTGGTGGACTCCTACATCAACATCGGGGCGCGCTGGTCGCTGGATCCCATGGACAACGTGAACCCCTTCAACCACCACCGCAATGCGGGGCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAATTTTTCGCCATCAAGAGCCTCCTGCTCCTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTGCAGAGCTCCCTCGGCAACGACCTGCGCACGGACGGGGCCTCCATCTCCTTCACCAGCATCAACCTCTACGCCACCTTCTTCCCCATGGCGCACAACACGGCCTCCACGCTCGAGGCCATGCTGCGCAACGACACCAACGACCAGTCCTTCAACGACTACCTCTCGGCGGCCAACATGCTCTACCCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCCGCCTTCCGCGGCTGGTCCTTCACGCGTCTCAAGACCAAGGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGTCTACTCGGGCTCCATCCCCTACCTCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACTCCTCCGTCAGCTGGCCCGGCAACGACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGGCTTCTACGTGCCCGAGGGCTACAAGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGGACGAGGTCAACTACAAGGACTACCAGGCCGTCACCCTGGCCTACCAGCACAACAACTCGGGCTTCGTCGGCTACCTCGCGCCCACCATGCGCCAGGGCCAGCCCTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTCACCAGCGTCACCCAGAAAAAGTTCCTCTGCGACAGGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCGACCTCGGCCAGAACATGCTCTATGCCAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCCACCCTTCTCTATGTTGTCTTCGAAGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATCGAGGCCGTCTACCTGCGCACCCCCTTCTCGGCCGGTAACGCCACCACCTAAGCTCTTGCTTCTTGCAAGCCATGGCCGCGGGCTCCGGCGAGCAGGAGCTCAGGGCCATCATCCGCGACCTGGGCTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGCTTCCCGGGATTCATGGCCCCGCACAAGCTGGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCACTGGCTGGCCTTCGCCTGGAACCCGCGCTCGAACACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACGAGCGCCTCAAGCAGATCTACCAGTTCGAGTACGAGGGCCTGCTGCGCCGCAGCGCCCTGGCCACCGAGGACCGCTGCGTCACCCTGGAAAAGTCCACCCAGACCGTGCAGGGTCCGCGCTCGGCCGCCTGCGGGCTCTTCTGCTGCATGTTCCTGCACGCCTTCGTGCACTGGCCCGACCGCCCCATGGACAAGAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACCCACCCTGCGCCGCAACCAGGAGGCGCTCTACCGCTTCCTCAACTCCCACTCCGCCTACTTTCGCTCCCACCGCGCGCGCATCGAGAAGGCCACCGCCTTCGACCGCATGAATCAAGACATGTAAACCGTGTGTGTATGTTAAATGTCTTTAATAAACAGCACTTTCATGTTACACATGCATCTGAGATGATTTATTTAGAAATCGAAAGGGTTCTGCCGGGTCTCGGCATGGCCCGCGGGCAGGGACACGTTGCGGAACTGGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTGGGCAGCGGGGTGTCGGGGAAGGAGTCGGTCCACAGCTTCCGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTGGGACCCGCGTTCTGCGCGCGGGAGTTGCGGTACACGGGGTTGCAGCACTGGAACACCATCAGGGCCGGGTGCTTCACGCTCGCCAGCACCGTCGCGTCGGTGATGCTCTCCACGTCGAGGTCCTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGGTCTGCCTTCCCATGGTGGGCACGCACCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGGTCGGCGTTCATCCCCGGGTACATGGCCTTCATGAAAGCCTCCAATTGCCTGAACGCCTGCTGGGCCTTGGCTCCCTCGGTGAAGAAGACCCCGCAGGACTTGCTAGAGAACTGGTTGGTGGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGTTGGCCAGCTGCACCACGCTGCGCCCCCAGCGGTTCTGGGTGATCTTGGCCCGGTCGGGGTTCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTCGCCACATCCATCTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCCCTCGGCCTCGGTGCACCCGTGCAGCCACAGCGCGCACCCGGTGCACTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCGTGCACGAAGCCCTGCAGGAAGCGGCCCATCATGGTGGTCAGGGTCTTGTTGCTAGTGAAGGTCAGCGGAATGCCGCGGTGCTCCTCGTTGATGTACAGGTGGCAGATGCGGCGGTACACCTCGCCCTGCTCGGGCATCAGCTGGAAGTTGGCTTTCAGGTCGGTCTCCACGCGGTAGCGGTCCATCAGCATAGTCATGATTTCCATACCCTTCTCCCAGGCCGAGACGATGGGCAGGCTCATAGGGTTCTTCACCATCATCTTAGCGCTAGCAGCCGCGGCCAGGGGGTCGCTCTCGTCCAGGGTCTCAAAGCTCCGCTTGCCGTCCTTCTCGGTGATCCGCACCGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTGTCTTTCGTCCTCGCTGTCCTGGCTGACGTCCTGCAGGACCACATGCTTGGTCTTGCGGGGTTTCTTCTTGGGCGGCAGCGGCGGCGGAGATGTTGGAGATGGCGAGGGGGAGCGCGAGTTCTCGCTCACCACTACTATCTCTTCCTCTTCTTGGTCCGAGGCCACGCGGCGGTAGGTATGTCTCTTCGGGGGCAGAGGCGGAGGCGACGGGCTCTCGCCGCCGCGACTTGGCGGATGGCTGGCAGAGCCCCTTCCGCGTTCGGGGGTGCGCTCCCGGCGGCGCTCTGACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAGGAACAACAAGCATGGAGACTCAGCCATCGCCAACCTCGCCATCTGCCCCCACCGCCGACGAGAAGCAGCAGCAGCAGAATGAAAGCTTAACCGCCCCGCCGCCCAGCCCCGCCACCTCCGACGCGGCCGTCCCAGACATGCAAGAGATGGAGGAATCCATCGAGATTGACCTGGGCTATGTGACGCCCGCGGAGCACGAGGAGGAGCTGGCAGTGCGCTTTTCACAAGAAGAGATACACCAAGAACAGCCAGAGCAGGAAGCAGAGAATGAGCAGAGTCAGGCTGGGCTCGAGCATGACGGCGACTACCTCCACCTGAGCGGGGGGGAGGACGCGCTCATCAAGCATCTGGCCCGGCAGGCCACCATCGTCAAGGATGCGCTGCTCGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGAGTTGAACCTCTTCTCGCCGCGCGTGCCCCCCAAGCGCCAGCCCAATGGCACCTGCGAGCCCAACCCGCGCCTCAACTTCTACCCGGTCTTCGCGGTGCCCGAGGCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAAGATCCCCGTCTCCTGCCGCGCCAACCGCACCCGCGCCGACGCCCTTTTCAACCTGGGTCCCGGCGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGTTCCCAAGATCTTCGAGGGTCTGGGCAGCGACGAGACTCGGGCCGCGAACGCTCTGCAAGGAGAAGGAGGAGAGCATGAGCACCACAGCGCCCTGGTCGAGTTGGAAGGCGACAACGCGCGGCTGGCGGTGCTCAAACGCACGGTCGAGCTGACCCATTTCGCCTACCCGGCTCTGAACCTGCCCCCCAAAGTCATGAGCGCGGTCATGGACCAGGTGCTCATCAAGCGCGCGTCGCCCATCTCCGAGGACGAGGGCATGCAAGACTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCCCGGTGGCTGGGTCCTAATGCTAGTCCCCAGAGTTTGGAAGAGCGGCGCAAACTCATGATGGCCGTGGTCCTGGTGACCGTGGAGCTGGAGTGCCTGCGCCGCTTCTTCGCCGACGCGGAGACCCTGCGCAAGGTCGAGGAGAACCTGCACTACCTCTTCAGGCACGGGTTCGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGGGGCAGAACGTGCTGCACACCACCCTGCGCGGGGAGGCCCGGCGCGACTACATCCGCGACTGCGTCTACCTCTACCTCTGCCACACCTGGCAGACGGGCATGGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTGAAAGAGCTCTGCAAGCTCCTGCAGAAGAACCTCAAGGGTCTGTGGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCCGACCTCATTTTCCCCGAGCGCCTCAGGCTGACGCTGCGCAACGGCCTGCCCGACTTTATGAGCCAAAGCATGTTGCAAAACTTTCGCTCTTTCATCCTCGAACGCTCCGGAATCCTGCCCGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCCCCCGCCGCTGTGGAGCCACTGCTACCTGCTGCGCCTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCAGCGGCGAGGGCCTGCTCGAGTGCCACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTGCTGAGCGAGACCCAGATCATCGGCACCTTCGAGTTGCAAGGGCCCAGCGAAGGCGAGGGTTCAGCCGCCAAGGGGGGTCTGAAACTCACCCCGGGGCTGTGGACCTCGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCTACGAGGACCAATCCCATCCGCCCAAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAAGCCATCCAGAAATCCCGCCAAGAATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTCGACCCCCAGACCGGTGAGGAGCTCAACCCCGGCTTCCCCCAGGATGCCCCGAGGAAACAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAGGAAGACTGGGAGAACAGCAGTCAGGCAGAGGAGGAGGAGATGGAGGAAGACTGGGACAGCACTCAGGCAGAGGAGGACAGCCTGCAAGACAGTCTGGAGGAAGACGAGGAGGAGGCAGAGGAGGAGGTGGAAGAAGCAGCCGCCGCCAGACCGTCGTCCTCGGCGGGGGAGAAAGCAAGCAGCACGGATACCATCTCCGCTCCGGGTCGGGGTCCCGCTCGACCACACAGTAGATGGGACGAGACCGGACGATTCCCGAACCCCACCACCCAGACCGGTAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTCCTGCTTGCAGGCCTGCGGGGGCAACATCTCCTTCACCCGGCGCTACCTGCTCTTCCACCGCGGGGTGAACTTTCCCCGCAACATCTTGCATTACTACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGCAGCAGCAGAAAAAGACCAGCAGAAAACCAGCAGCTAGAAAATCCACAGCGGCGGCAGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAAACCCGGGAGCTGAGGAACCGGATCTTTCCCACCCTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTGCGCTCGCTCACCCGCAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACTCTCGAGGACGCCGAGGCTCTCTTCAACAAGTACTGCGCGCTCACTCTTAAAGAGTAGCCCGCGCCCGCCCAGTCGCAGAAAAAGGCGGGAATTACGTCACCTGTGCCCTTCGCCCTAGCCGCCTCCACCCATCATCATGAGCAAAGAGATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGGCCGCCGGTGCCGCCCAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGCGATGATCTCACGGGTGAATGACATCCGCGCCCACCGAAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAATTGGCCCGCCGCCCTGGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCCAGCTGACTAACTCAGGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCGCCCCGCTCAGGGTATAAAGCGGCTGGTGATCCGGGGCAGAGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCGCTGGGTCTGCGACCTGACGGAGTCTTCCAACTCGCCGGATCGGGGAGATCTTCCTTCACGCCTCGTCAGGCCGTCCTGACTTTGGAGAGTTCGTCCTCGCAGCCCCGCTCGGGTGGCATCGGCACTCTCCAGTTCGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGCTCCCCCGGCCACTACCCGGACGAGTTCATCCCGAACTTCGACGCCATCAGCGAGTCGGTGGACGGCTACGATTGAAACTAATCACCCCCTTATCCAGTGAAATAAAGATCATATTGATGATGATTTTACAGAAATAAAAAATAATCATTTGATTTGAAATAAAGATACAATCATATTGATGATTTGAGTTTAACAAAAAAATAAAGAATCACTTACTTGAAATCTGATACCAGGTCTCTGTCCATGTTTTCTGCCAACACCACTTCACTCCCCTCTTCCCAGCTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACGCTGAAGGGGATGTCAAATTCCTCCTGTCCCTCAATCTTCATTTTATCTTCTATCAGATGTCCAAAAAGCGCGTCCGGGTGGATGATGACTTCGACCCCGTCTACCCCTACGATGCAGACAACGCACCGACCGTGCCCTTCATCAACCCCCCCTTCGTCTCTTCAGATGGATTCCAAGAGAAGCCCCTGGGGGTGTTGTCCCTGCGACTGGCCGACCCCGTCACCACCAAGAACGGGGAAATCACCCTCAAGCTGGGAGAGGGGGTGGACCTCGATTCCTCGGGAAAACTCATCTCCAACACGGCCACCAAGGCCGCCGCCCCTCTCAGTTTTTCCAACAACACCATTTCCCTTAACATGGATCACCCCTTTTACACTAAAGATGGAAAATTATCCTTACAAGTTTCTCCACCATTAAATATACTGAGAACAAGCATTCTAAACACACTAGCTTTAGGTTTTGGATCAGGTTTAGGACTCCGTGGCTCTGCCTTGGCAGTACAGTTAGTCTCTCCACTTACATTTGATACTGATGGAAACATAAAGCTTACCTTAGACAGAGGTTTGCATGTTACAACAGGAGATGCAATTGAAAGCAACATAAGCTGGGCTAAAGGTTTAAAATTTGAAGATGGAGCCATAGCAACCAACATTGGAAATGGGTTAGAGTTTGGAAGCAGTAGTACAGAAACAGGTGTTGATGATGCTTACCCAATCCAAGTTAAACTTGGATCTGGCCTTAGCTTTGACAGTACAGGAGCCATAATGGCTGGTAACAAAGAAGACGATAAACTCACTTTGTGGACAACACCTGATCCATCACCAAACTGTCAAATACTCGCAGAAAATGATGCAAAACTAACACTTTGCTTGACTAAATGTGGTAGTCAAATACTGGCCACTGTGTCAGTCTTAGTTGTAGGAAGTGGAAACCTAAACCCCATTACTGGCACCGTAAGCAGTGCTCAGGTGTTTCTACGTTTTGATGCAAACGGTGTTCTTTTAACAGAACATTCTACACTAAAAAAATACTGGGGGTATAGGCAGGGAGATAGCATAGATGGCACTCCATATACCAATGCTGTAGGATTCATGCCCAATTTAAAAGCTTATCCAAAGTCACAAAGTTCTACTACTAAAAATAATATAGTAGGGCAAGTATACATGAATGGAGATGTTTCAAAACCTATGCTTCTCACTATAACCCTCAATGGTACTGATGACAGCAACAGTACATATTCAATGTCATTTTCATACACCTGGACTAATGGAAGCTATGTTGGAGCAACATTTGGGGCTAACTCTTATACCTTCTCATACATCGCCCAAGAATGAACACTGTATCCCACCCTGCATGCCAACCCTTCCCACCCCACTCTGTGGAACAAACTCTGAAACACAAAATAAAATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTACAGGATTCGAGCAGTTATTTTTCCTCCACCCTCCCAGGACATGGAATACACCACCCTCTCCCCCCGCACAGCCTTGAACATCTGAATGCCATTGGTGATGGACATGCTTTTGGTCTCCACGTTCCACACAGTTTCAGAGCGAGCCAGTCTCGGGTCGGTCAGGGAGATGAAACCCTCCGGGCACTCCCGCATCTGCACCTCACAGCTCAACAGCTGAGGATTGTCCTCGGTGGTCGGGATCACGGTTATCTGGAAGAAGCAGAAGAGCGGCGGTGGGAATCATAGTCCGCGAACGGGATCGGCCGGTGGTGTCGCATCAGGCCCCGCAGCAGTCGCTGCCGCCGCCGCTCCGTCAAGCTGCTGCTCAGGGGGTCCGGGTCCAGGGACTCCCTCAGCATGATGCCCACGGCCCTCAGCATCAGTCGTCTGGTGCGGCGGGCGCAGCAGCGCATGCGGATCTCGCTCAGGTCGCTGCAGTACGTGCAACACAGAACCACCAGGTTGTTCAACAGTCCATAGTTCAACACGCTCCAGCCGAAACTCATCGCGGGAAGGATGCTACCCACGTGGCCGTCGTACCAGATCCTCAGGTAAATCAAGTGGTGCCCCCTCCAGAACACGCTGCCCACGTACATGATCTCCTTGGGCATGTGGCGGTTCACCACCTCCCGGTACCACATCACCCTCTGGTTGAACATGCAGCCCCGGATGATCCTGCGGAACCACAGGGCCAGCACCGCCCCGCCCGCCATGCAGCGAAGAGACCCCGGGTCCCGGCAATGGCAATGGAGGACCCACCGCTCGTACCCGTGGATCATCTGGGAGCTGAACAAGTCTATGTTGGCACAGCACAGGCATATGCTCATGCATCTCTTCAGCACTCTCAACTCCTCGGGGGTCAAAACCATATCCCAGGGCACGGGGAACTCTTGCAGGACAGCGAACCCCGCAGAACAGGGCAATCCTCGCACAGAACTTACATTGTGCATGGACAGGGTATCGCAATCAGGCAGCACCGGGTGATCCTCCACCAGAGAAGCGCGGGTCTCGGTCTCCTCACAGCGTGGTAAGGGGGCCGGCCGATACGGGTGATGGCGGGACGCGGCTGATCGTGTTCGCGACCGTGTCATGATGCAGTTGCTTTCGGACATTTTCGTACTTGCTGTAGCAGAACCTGGTCCGGGCGCTGCACACCGATCGCCGGCGGCGGTCTCGGCGCTTGGAACGCTCGGTGTTGAAATTGTAAAACAGCCACTCTCTCAGACCGTGCAGCAGATCTAGGGCCTCAGGAGTGATGAAGATCCCATCATGCCTGATGGCTCTGATCACATCGACCACCGTGGAATGGGCCAGACCCAGCCAGATGATGCAATTTTGTTGGGTTTCGGTGACGGCGAGCCTCGGGAACAACGATGAAGTAAATGCAAGCGGTGCGTTCCAGCATGGTTAGTTAGCTGATCTGTAGAAAAAACAAAAATGAACATTAAACCATGCTAGCCTGGCGAACAGGTGGGTAAATCGTTCTCTCCAGCACCAGGCAGGCCACGGGGTCTCCGGCGCGACCCTCGTAAAAATTGTCGCTATGATTGAAAACCATCACAGAGAGACGTTCCCGGTGGCCGGCGTGAATGATTCGACAAGATGAATACACCCCCGGAACATTGGCGTCCGCGAGTGAAAAAAAGCGCCCGAGGAAGCAATAAGGCACTACAATGCTCAGTCTCAAGTCCAGCAAAGCGATGCCATGCGGATGAAGCACAAAATTCTCAGGTGCGTACAAAATGTAATTACTCCCCTCCTGCACAGGCAGCAAAGCCCCCGATCCCTCCAGGTACACATACAAAGCCTCAGCGTCCATAGCTTACCGAGCAGCAGCACACAACAGGCGCAAGAGTCAGAGAAAGGCTGAGCTCTAACCTGTCCACCCGCTCTCTGCTCAATATATAGCCCAGATCTACACTGACGTAAAGGCCAAAGTCTAAAAATACCCGCCAAATAATCACACACGCCCAGCACACGCCCAGAAACCGGTGACACACTCAAAAAAATACGCGCACTTCCTCAAACGCCCAAAACTGCCGTCATTTCCGGGTTCCCACGCTACGTCATCAAAACACGACTTTCAAATTCCGTCGACCGTTAAAAACGTCACCCGCCCCGCCCCTAACGGTCGCCCGTCTCTCAGCCAATCAGCGCCCCGCATCCCCAAATTCAAACACCTCATTTGCATATTAACGCGCACAAAAAGTTTGAGGTATATTATTGATGATG

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1-145. (canceled)
 146. An isolated cell comprising an adenovirus vector,wherein the adenovirus vector comprises: an adenoviral backbonecomprising one or more genes or regulatory sequences of an adenovirusgenome, wherein the adenoviral backbone comprises a partially deleted E4gene with reference to the adenovirus genome, wherein the partiallydeleted E4 gene comprises a partially-deleted E4orf2 region, a deletedE4orf3 region, and a partially-deleted E4orf4 region, and wherein theadenovirus vector further comprises a cassette, the cassette comprising:(1) at least one payload nucleic acid sequence, optionally wherein theat least one payload nucleic acid sequence encodes a polypeptide,optionally wherein the polypeptide comprises an antigen, optionallywherein the antigen comprises: (a) a MHC class I epitope, (b) a MHCclass II epitope, (c) an epitope capable of stimulating a B cellresponse, or (d) a combination thereof, and optionally wherein the atleast one payload nucleic acid sequence further comprises a 5′ linkersequence and/or a 3′ linker sequence, and optionally wherein; (2) atleast one promoter sequence operably linked to the at least one payloadnucleic acid sequence, (3) optionally, at least one universal MHC classII antigen-encoding nucleic acid sequence; (4) optionally, at least oneGPGPG-encoding linker sequence (SEQ ID NO:56); and (5) optionally, atleast one polyadenylation sequence.
 147. The cell of claim 146, whereinthe isolated cell is selected from the group consisting of: a CHO cell,a HEK293 cell or variants thereof, a 911 cell, a HeLa cell, a A549 cell,a LP-293 cell, a PER.C6 cell, and a AE1-2a cell.
 148. The cell of claim146, wherein the isolated cell is a HEK293 cell or variants thereof.149. The cell of claim 148, wherein the HEK293 cell variant comprises anadenoviral E1 gene, optionally wherein the adenoviral E1 gene is stablyintegrated.
 150. The cell of claim 146, wherein: (a) the partiallydeleted E4 gene comprises the E4 gene sequence shown in SEQ ID NO:1except for lacking the partially-deleted E4orf2 region, the deletedE4orf3 region, and the partially-deleted E4orf4 region; and (b) the oneor more genes or regulatory sequences of the adenovirus genome compriseone or more genes or regulatory sequences of the ChAdV68 sequence shownin SEQ ID NO:1, optionally wherein the one or more genes or regulatorysequences comprise at least one of the chimpanzee adenovirus invertedterminal repeat (ITR), E1A, E1B, E2A, E2B, E3, L1, L2, L3, L4, and L5genes of the sequence shown in SEQ ID NO:1.
 151. The cell of claim 150,wherein the one or more genes or regulatory sequences of the ChAdV68sequence shown in SEQ ID NO:1 comprise: A) nucleotides 2 to 34,915 ofthe sequence shown in SEQ ID NO:1, or B) nucleotides 2 to 34,915 of thesequence shown in SEQ ID NO:1 except for lacking: (i) nucleotidescorresponding to a deletion in the E1 gene shown in SEQ ID NO:1; and/or(ii) nucleotides corresponding to a deletion in the E3 gene shown in SEQID NO:1.
 152. The cell of claim 150, wherein the partially-deletedE4orf2 region, the deleted E4orf3 region, and the partially-deletedE4orf4 region is a deletion of nucleotides about in the range of 34,916to 35,642 of the sequence shown in SEQ ID NO:1.
 153. The cell of claim150, wherein the partially-deleted E4orf2 region, the deleted E4orf3region, and the partially-deleted E4orf4 region is a deletion ofnucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1. 154.The cell of claim 150, wherein the adenoviral backbone comprises: A) atleast nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO:1,except for lacking nucleotides 34,916 to 35,642 of the sequence shown inSEQ ID NO:1 corresponding to the partially deleted E4 gene, B) at leastnucleotides 2 to 36,518 of the sequence shown in SEQ ID NO:1, except forlacking nucleotides 34,916 to 35,642 corresponding to the partiallydeleted E4 gene and lacking nucleotides 577 to 3403 of the sequenceshown in SEQ ID NO:1, C) at least nucleotides 2 to 36,518 of thesequence shown in SEQ ID NO:1, except for lacking nucleotides 34,916 to35,642 corresponding to the partially deleted E4 gene and lackingnucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1, or D)at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO:1,except for lacking nucleotides 34,916 to 35,642 corresponding to thepartially deleted E4 gene, lacking nucleotides 577 to 3403, and lackingnucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1. 155.The cell of claim 146, wherein the cassette is inserted in the vector atthe E1 region, E3 region, and/or any deleted AdV region that allowsincorporation of the cassette.
 156. The cell of claim 146, wherein atleast one of the at least one payload nucleic acid sequences encodes apolypeptide sequence capable of undergoing antigen processing into anepitope, wherein the epitope is known or suspected to be presented byMHC class I on a surface of a cell, and wherein the surface of the cellis a tumor cell surface.
 157. The cell of claim 146, wherein at leastone of the at least one payload nucleic acid sequences encodes anepitope with at least one alteration that makes the encoded epitopesequence distinct from the corresponding peptide sequence encoded by awild-type nucleic acid sequence, optionally wherein the at least onealteration comprises a point mutation, a frameshift mutation, anon-frameshift mutation, a deletion mutation, an insertion mutation, asplice variant, a genomic rearrangement, or a proteasome-generatedspliced antigen.
 158. The cell of claim 146, wherein one or more of theat least one payload nucleic acid sequences encode an MHC Iepitope-encoding nucleic acid sequence inclusive of the optional 5′linker sequence and the optional 3′ linker sequences that encodes apeptide 25 amino acids in length.
 159. The cell of claim 146, wherein atleast one of the at least one payload nucleic acid sequences is linkedto a distinct payload nucleic acid sequence with a nucleic acid sequenceencoding a linker.
 160. The cell of claim 159, wherein the linkercomprises one or more native sequences flanking the antigen derived fromthe cognate protein of origin and that is at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acidresidues in length.
 161. The cell of claim 146, wherein the adenovirusvector comprises: A) a modified ChAdV68 sequence, wherein the modifiedChAdV68 sequence comprises: (i) the partially deleted E4 gene comprisingthe E4 gene sequence shown in SEQ ID NO:1 except for lacking nucleotides34,916 to 35,642 of the sequence shown in SEQ ID NO:1; (ii) (1)nucleotides 2 to 34,915 of the sequence shown in SEQ ID NO:1 or (ii) (2)nucleotides 2 to 34,915 of the sequence shown in SEQ ID NO:1 except forlacking, a) nucleotides 577 to 3403 of the sequence shown in SEQ ID NO:1corresponding to an E1 deletion; and/or b) nucleotides 27,125 to 31,825of the sequence shown in SEQ ID NO:1 corresponding to an E3 deletionwherein the partially deleted E4 gene is 3′ of the nucleotide 34,915 ofthe sequence shown in SEQ ID NO:1; and (iii) nucleotides 35,643 to36,518 of the sequence shown in SEQ ID NO:1, wherein the partiallydeleted E4 gene is 5′ of the nucleotide 35,643 of the sequence shown inSEQ ID NO:1; B) a CMV-derived promoter sequence; C) an SV40polyadenylation signal nucleotide sequence; and D) a cassette, thecassette comprising at least one at least one payload nucleic acidsequence encoding: (i) at least one MHC class I epitope, optionallywherein the at least one MHC class I epitope comprises at least 2distinct MHC class I epitopes linearly linked to each other and eachoptionally comprising: (A) at least one alteration that makes theencoded peptide sequence distinct from the corresponding peptidesequence encoded by a wild-type nucleic acid sequence, wherein thedistinct MHC I epitope is 7-15 amino acids in length, (B) a native 5′linker sequence that encodes a native N-terminal amino acid sequence ofthe epitope, and wherein the 5′ linker sequence encodes a peptide thatis between 2-20 amino acids in length, (C) a native 3′ linker sequencethat encodes a native C-terminal amino acid sequence of the epitope, andwherein the 3′ linker sequence encodes a peptide that is between 2-20amino acids in length, or (D) combinations thereof, (ii) at least oneMHC class II epitope, optionally wherein the at least one MHC class IIepitope comprises at least 2 distinct MHC class II epitopes, (iii) atleast one an epitope capable of stimulating a B cell response, or (iv)combinations thereof, and wherein the cassette is inserted within adeleted region of ChAdV68 and the CMV-derived promoter sequence isoperably linked to the cassette.
 162. The cell of claim 146, wherein theat least one promoter sequence is a regulatable promoter comprising atetracycline (TET) repressor protein (TETr) controlled promoter, andwherein the isolated cell is engineered to engineered to express theTETr protein.
 163. The cell of claim 146, wherein at least one of the atleast one payload nucleic acid sequences encodes: (a) an infectiousdisease organism peptide selected from the group consisting of: apathogen-derived peptide, a virus-derived peptide, a bacteria-derivedpeptide, a fungus-derived peptide, and a parasite-derived peptide; (b)an epitope with at least one alteration that makes the encoded epitopesequence distinct from the corresponding peptide sequence encoded by awild-type nucleic acid sequence; (c) a polypeptide sequence or portionthereof comprising an epitope capable of stimulating a B cell response,wherein the polypeptide sequence or portion thereof comprises afull-length protein, a protein domain, a protein subunit, or anantigenic fragment predicted or known to be capable of being bound by anantibody; and/or (d) a non-coding nucleic acid sequence, optionallywherein the non-coding nucleic acid sequence comprises an RNAinterference (RNAi) polynucleotide or genome-editing systempolynucleotide.
 164. A method of manufacturing an adenovirus vector, themethod comprising: (A) one or both of (i) transfecting a plasmidsequence comprising the adenovirus vector into a host cell, and (ii)infecting a host cell with an adenovirus comprising the adenovirusvector, wherein the adenovirus vector comprises: an adenoviral backbonecomprising one or more genes or regulatory sequences of an adenovirusgenome, wherein the adenoviral backbone comprises a partially deleted E4gene with reference to the adenovirus genome, wherein the partiallydeleted E4 gene comprises a partially-deleted E4orf2 region, a deletedE4orf3 region, and a partially-deleted E4orf4 region, and optionally,wherein the adenovirus vector further comprises a cassette, the cassettecomprising: (1) at least one payload nucleic acid sequence, optionallywherein the at least one payload nucleic acid sequence encodes apolypeptide, optionally wherein the polypeptide comprises an antigen,optionally wherein the antigen comprises: (a) a MHC class I epitope, (b)a MHC class II epitope, (c) an epitope capable of stimulating a B cellresponse, or (d) a combination thereof, and optionally wherein the atleast one payload nucleic acid sequence further comprises a 5′ linkersequence and/or a 3′ linker sequence, and optionally wherein; (2) atleast one promoter sequence operably linked to the at least one payloadnucleic acid sequence, (3) optionally, at least one universal MHC classII antigen-encoding nucleic acid sequence; (4) optionally, at least oneGPGPG-encoding linker sequence (SEQ ID NO:56); and (5) optionally, atleast one polyadenylation sequence; and (B) isolating the adenovirusvector from the host cell.
 165. The method of manufacturing of claim164, wherein the isolating comprises lysing the host cell to obtain acell lysate comprising the adenovirus vector.
 166. The method ofmanufacturing of claim 165, wherein the isolating further comprisespurifying the adenovirus vector from the cell lysate.
 167. The method ofmanufacturing of claim 166, wherein the purifying comprises one or moreof chromatographic separation, centrifugation, virus precipitation, andfiltration.
 168. The method of manufacturing of claim 164, wherein thehost cell is selected from the group consisting of: a CHO cell, a HEK293cell or variants thereof, a 911 cell, a HeLa cell, a A549 cell, a LP-293cell, a PER.C6 cell, and a AE1-2a cell.
 169. The method of manufacturingof claim 164, wherein: (a) the partially deleted E4 gene comprises theE4 gene sequence shown in SEQ ID NO:1 except for lacking thepartially-deleted E4orf2 region, the deleted E4orf3 region, and thepartially-deleted E4orf4 region; and (b) the one or more genes orregulatory sequences of the adenovirus genome comprise one or more genesor regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO:1,optionally wherein the one or more genes or regulatory sequencescomprise at least one of the chimpanzee adenovirus inverted terminalrepeat (ITR), E1A, E1B, E2A, E2B, E3, L1, L2, L3, L4, and L5 genes ofthe sequence shown in SEQ ID NO:1.
 170. The method of manufacturing ofclaim 164, wherein at least one of the at least one payload nucleic acidsequences encodes: (a) an infectious disease organism peptide selectedfrom the group consisting of: a pathogen-derived peptide, avirus-derived peptide, a bacteria-derived peptide, a fungus-derivedpeptide, and a parasite-derived peptide; (b) an epitope with at leastone alteration that makes the encoded epitope sequence distinct from thecorresponding peptide sequence encoded by a wild-type nucleic acidsequence; (c) a polypeptide sequence or portion thereof comprising anepitope capable of stimulating a B cell response, wherein thepolypeptide sequence or portion thereof comprises a full-length protein,a protein domain, a protein subunit, or an antigenic fragment predictedor known to be capable of being bound by an antibody; and/or (d) anon-coding nucleic acid sequence, optionally wherein the non-codingnucleic acid sequence comprises an RNA interference (RNAi)polynucleotide or genome-editing system polynucleotide.
 171. The methodof manufacturing of claim 169, wherein the one or more genes orregulatory sequences of the ChAdV68 sequence shown in SEQ ID NO:1comprise: A) nucleotides 2 to 34,915 of the sequence shown in SEQ IDNO:1, or B) nucleotides 2 to 34,915 of the sequence shown in SEQ ID NO:1except for lacking: (i) nucleotides corresponding to a deletion in theE1 gene shown in SEQ ID NO:1; and/or (ii) nucleotides corresponding to adeletion in the E3 gene shown in SEQ ID NO:1.
 172. The method ofmanufacturing of claim 169, wherein the partially-deleted E4orf2 region,the deleted E4orf3 region, and the partially-deleted E4orf4 region is adeletion of nucleotides about in the range of 34,916 to 35,642 of thesequence shown in SEQ ID NO:1.
 173. The method of manufacturing of claim169, wherein the partially-deleted E4orf2 region, the deleted E4orf3region, and the partially-deleted E4orf4 region is a deletion ofnucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1. 174.The method of manufacturing of claim 169, wherein the adenoviralbackbone comprises: A) at least nucleotides 2 to 36,518 of the sequenceshown in SEQ ID NO:1, except for lacking nucleotides 34,916 to 35,642 ofthe sequence shown in SEQ ID NO:1 corresponding to the partially deletedE4 gene, B) at least nucleotides 2 to 36,518 of the sequence shown inSEQ ID NO:1, except for lacking nucleotides 34,916 to 35,642corresponding to the partially deleted E4 gene and lacking nucleotides577 to 3403 of the sequence shown in SEQ ID NO:1, C) at leastnucleotides 2 to 36,518 of the sequence shown in SEQ ID NO:1, except forlacking nucleotides 34,916 to 35,642 corresponding to the partiallydeleted E4 gene and lacking nucleotides 27,125 to 31,825 of the sequenceshown in SEQ ID NO:1, or D) at least nucleotides 2 to 36,518 of thesequence shown in SEQ ID NO:1, except for lacking nucleotides 34,916 to35,642 corresponding to the partially deleted E4 gene, lackingnucleotides 577 to 3403, and lacking nucleotides 27,125 to 31,825 of thesequence shown in SEQ ID NO:1.
 175. The method of manufacturing of claim164, wherein the cassette is inserted in the vector at the E1 region, E3region, and/or any deleted AdV region that allows incorporation of thecassette.
 176. The method of manufacturing of claim 164, wherein atleast one of the at least one payload nucleic acid sequences encodes apolypeptide sequence capable of undergoing antigen processing into anepitope, wherein the epitope is known or suspected to be presented byMHC class I on a surface of a cell, and wherein the surface of the cellis a tumor cell surface.
 177. The method of manufacturing of claim 164,wherein one or more of the at least one payload nucleic acid sequencesencode an MHC I epitope-encoding nucleic acid sequence inclusive of theoptional 5′ linker sequence and the optional 3′ linker sequences thatencodes a peptide 25 amino acids in length.
 178. The method ofmanufacturing of claim 164, wherein at least one of the at least onepayload nucleic acid sequences is linked to a distinct payload nucleicacid sequence with a nucleic acid sequence encoding a linker.
 179. Themethod of manufacturing of claim 178, wherein the linker comprises oneor more native sequences flanking the antigen derived from the cognateprotein of origin and that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues inlength.
 180. The method of manufacturing of claim 164, wherein theadenovirus vector comprises: A) a modified ChAdV68 sequence, wherein themodified ChAdV68 sequence comprises: (i) the partially deleted E4 genecomprising the E4 gene sequence shown in SEQ ID NO:1 except for lackingnucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1; (ii)(1) nucleotides 2 to 34,915 of the sequence shown in SEQ ID NO:1 or (ii)(2) nucleotides 2 to 34,915 of the sequence shown in SEQ ID NO:1 exceptfor lacking, a) nucleotides 577 to 3403 of the sequence shown in SEQ IDNO:1 corresponding to an E1 deletion; and/or b) nucleotides 27,125 to31,825 of the sequence shown in SEQ ID NO:1 corresponding to an E3deletion wherein the partially deleted E4 gene is 3′ of the nucleotide34,915 of the sequence shown in SEQ ID NO:1; and (iii) nucleotides35,643 to 36,518 of the sequence shown in SEQ ID NO:1, wherein thepartially deleted E4 gene is 5′ of the nucleotide 35,643 of the sequenceshown in SEQ ID NO:1; B) a CMV-derived promoter sequence; C) an SV40polyadenylation signal nucleotide sequence; and D) a cassette, thecassette comprising at least one at least one payload nucleic acidsequence encoding: (i) at least one MHC class I epitope, optionallywherein the at least one MHC class I epitope comprises at least 2distinct MHC class I epitopes linearly linked to each other and eachoptionally comprising: (A) at least one alteration that makes theencoded peptide sequence distinct from the corresponding peptidesequence encoded by a wild-type nucleic acid sequence, wherein thedistinct MHC I epitope is 7-15 amino acids in length, (B) a native 5′linker sequence that encodes a native N-terminal amino acid sequence ofthe epitope, and wherein the 5′ linker sequence encodes a peptide thatis between 2-20 amino acids in length, (C) a native 3′ linker sequencethat encodes a native C-terminal amino acid sequence of the epitope, andwherein the 3′ linker sequence encodes a peptide that is between 2-20amino acids in length, or (D) combinations thereof, (ii) at least oneMHC class II epitope, optionally wherein the at least one MHC class IIepitope comprises at least 2 distinct MHC class II epitopes, (iii) atleast one an epitope capable of stimulating a B cell response, or (iv)combinations thereof, and wherein the cassette is inserted within adeleted region of ChAdV68 and the CMV-derived promoter sequence isoperably linked to the cassette.
 181. The method of manufacturing ofclaim 164, wherein the at least one promoter sequence is a regulatablepromoter comprising a tetracycline (TET) repressor protein (TETr)controlled promoter, and wherein the host cell is engineered toengineered to express the TETr protein.